Agonist antibodies against the flk2/flt3 receptor and uses thereof

ABSTRACT

Agonist antibodies are disclosed which bind to the extracellular domain of the flk2/flt3 receptor and thereby activate the intracellular kinase domain thereof. The labeled antibodies are useful as diagnostics for detecting the presence of the flk2/flt3 receptor in primitive hematopoietic cells for example. The antibodies are able to cause primitive hematopoietic cells to proliferate and/or differentiate and thereby enhance repopulation of mature blood cell lineages in a mammal which has undergone chemo- or radiation therapy or bone marrow transplantation. The antibodies are further useful for treating mammals which have suffered a decrease in blood cells as a consequence of disease or a hemorrhage, for example.

This is a divisional of application Ser. No. 08/222,299 filed on Apr. 4,1994 now patented, U.S. Pat No. 5,635,388 which application isincorporated herein by reference and to which application prioity isclaimed under 35 USC §120.

FIELD OF THE INVENTION

This application relates to agonist antibodies against the flk2/flt3receptor and uses thereof. In particular, the invention relates to theuse of the antibodies for enhancing the proliferation and/ordifferentiation of primitive hematopoietic cells.

BACKGROUND OF THE INVENTION

A. Hematopoiesis

The process of blood cell formation whereby red and white blood cellsare replaced through the division of cells located in the bone marrow iscalled hematopoiesis. For a review of hematopoiesis see Dexter andSpooncer (Ann. Rev. Cell Biol., 3:423-441 [1987]).

There are many different types of blood cells which belong to distinctcell lineages. Along each lineage, there are cells at different stagesof maturation. Mature blood cells are specialized for differentfunctions. For example, erythrocytes are involved in O₂ and CO₂transport; T and B lymphocytes are involved in cell and antibodymediated immune responses, respectively; platelets are required forblood clotting; and the granulocytes and macrophages act as generalscavengers and accessory cells. Granulocytes can be further divided intobasophils, eosinophils, neutrophils and mast cells.

Each of the various blood cell types arises from pluripotent ortotipotent stem cells which are able to undergo self-renewal or giverise to progenitor cells or Colony Forming Units (CFU) that yield a morelimited array of cell types. As stem cells progressively lose theirability to self-renew, they become increasingly lineage restricted. Ithas been shown that stem cells can develop into multipotent cells(called "CFC-Mix" by Dexter and Spooncer, supra). Some of the CFC-Mixcells can undergo renewal whereas others lead to lineage-restrictedprogenitors which eventually develop into mature myeloid cells (e.g.,neutrophils, megakaryocytes, macrophages, basophils and erythroidcells). Similarly, pluripotent stem cells are able to give rise to PreBand PreT lymphoid cell lineages which differentiate into mature B and Tlymphocytes, respectively. Progenitors are defined by their progeny,e.g., granulocyte/macrophage colony-forming progenitor cells (GM-CFU)differentiate into neutrophils or macrophages; primitive erythroidburst-forming units (BFU-E) differentiate into erythroid colony-formingunits (CFU-E) which give rise to mature erythrocytes. Similarly, theMeg-CFU, Eos-CFU and Bas-CFU progenitors are able to differentiate intomegakaryocytes, eosinophils and basophils, respectively.

The number of pluripotent stem cells in the bone marrow is extremely lowand has been estimated to be in the order of about one per 10,000 to oneper 100,000 cells (Boggs et al., J. Clin. Inv., 70:242 [1982] andHarrison et al., PNAS, 85; 822 [1988]). Accordingly, characterization ofstem cells has been difficult. Therefore, various protocols forenriching pluripotent stem cells have been developed. See, for example,Matthews et al., Cell, 65:1143-1152 [1991]; WO 94/02157; Orlic et al.,Blood, 82(3):762-770 [1993]; and Visser et al., Stem Cells, 11(2):49-55[1993].

Various lineage-specific factors have been demonstrated to control cellgrowth, differentiation and the functioning of hematopoietic cells.These factors or cytokines include the interleukins (e.g., IL-3),granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophagecolony-stimulating factor (M-CSF), granulocyte colony-stimulating factor(M-CSF), erythropoietin (Epo), lymphotoxin, steel factor (SLF), tumornecrosis factor (TNF) and gamma-interferon. These growth factors have abroad spectrum of activity, from generalized to lineage-specific rolesin hematopoiesis, or a combination of both. For example, IL-3 appears toact on multipotent stem cells as well as progenitors restricted to thegranulocyte/macrophage, eosinophil, megakaryocyte, erythroid or mastcell lineages. On the other hand, Epo generally acts on fairly matureerythroid progenitor cells.

B. Tyrosine Kinases

Many cytokines involved in hematopoietic development stimulate receptorprotein tyrosine kinases (pTKs). For example, the c-kit pTK and itscognate ligand (KL) have been shown to play a role in hematopoiesis.Tyrosine kinases catalyze protein phosphorylation using tyrosine as asubstrate for phosphorylation. Members of the tyrosine kinase family canbe recognized by the presence of several conserved amino acid regions inthe tyrosine kinase catalytic domain (Hanks et al., Science, 241:42-52[1988]). Receptor protein tyrosine kinases share a similar architecture,with an intracellular catalytic portion, a hydrophobic transmembranedomain and an extracellular ligand-binding domain. The extracellulardomains (ECDs), which are responsible for ligand binding andtransmission of biological signals, have been shown to be composed of anumber of distinct structural motifs. The intracellular domain comprisesa catalytic protein tyrosine kinase. The binding of ligand to theextracellular portion is believed to promote dimerization of the pTKresulting in transphosphorylation and activation of the intracellulartyrosine kinase domain (see Schlessinger et al., Neuron, 9:383-391[1992]).

C. Flk2/Flt3 Receptor

A murine gene encoding a pTK which is expressed in cell populationsenriched for stem cells and primitive uncommitted progenitors has beenidentified and is called "fetal liver kinase-2" or "flk-2" by Matthewset al. in Cell, 65:1143-52 [1991]. Rosnet et al. independentlyidentified partial cDNA sequences for the same gene, which they call"flt3", from murine and human tissues (Genomics, 9:380-385 [1991]). Thefull length flt3 sequence has been published by Rosnet et al. inOncogene, 6:1641-1650 [1991]. The sequence for human flk2 is disclosedin WO 93/10136. Kuczynski et al. refer to a gene called "STK-1" which issaid to be the human homologue of murine flk2/flt3 (Blood, 82(10):PA486[1993]).

Matthews et al. isolated the flk2 cDNA from stem cell-enrichedhematopoietic tissue. In order to enrich for stem cells, murine fetalliver cells were fractionated using the AA4.1 monoclonal antibody and acocktail of antibodies raised against specific differentiation antigens,collectively called "Lin". Flk-2 was found to be expressed in AA4⁺, AA4⁺Lin^(lo) and AA4⁺ Lin^(br) cells, but not in AA4⁻ cells. The AA4⁺Lin^(lo) population contained all of the long-term pluripotent stemcells. The AA4⁺ Lin^(br) population was depleted of pluripotent stemcells but contained multipotent progenitors. The AA4⁻ population wasdevoid of primitive clonogenic cells but contained less primitiveprogenitors such as the CFU-E. Expression of flk2 in AA4⁺ Sca⁺ and AA4⁺Sca⁺ Lin^(lo) populations, which are considered to be highly enrichedstem cell populations, was further demonstrated. Additional expressionof flk2 in the day 14 thymus (at which stage the thymocyte population ishighly enriched in primitive precursors) was studied. Flk2 mRNA wasexpressed in the most immature T lymphocyte population (CD4⁻ 8⁻Thy-l^(lo) /IL-2R⁻). Overall, the results of the experiments describedin Matthews et al. indicate that flk2 is expressed in the most primitivestem/progenitor hematopoietic cells.

Poly(A)⁺ RNA expression in fetal and adult tissues was also investigatedby Matthews et al. Expression of flk2 mRNA in the fetal brain and liveras well as adult brain and bone marrow tissues was observed. Rosnet etal. similarly observed that the flt3 gene is expressed in placenta andin various adult tissues including gonads and the brain as well ashematopoietic cells (Oncogene, 6:1641-1650 [1991]). The flt3 transcriptidentified by Rosnet et al. was 3.7 kb long, except in the testis, wheretwo shorter transcripts were identified.

Small et al. (USA PNAS, 91:459-463 [1994]) have shown that antisenseoligonucleotides directed against the human homologue of the flk2/flt3gene inhibit colony formation in long term bone marrow cultures, whichresults further indicate that flk2/flt3 may transduce growth signals inhematopoietic stem cells.

WO 94/01576 refers to a soluble form of the flk2/flt3 receptor,designated flk-2ws, encoded by a 1.9 kb DNA fragment.

Dosil et al. prepared a chimeric receptor which consisted of theextracellular ligand-binding domain of the human fms pTK and thetransmembrane and tyrosine kinase domains of murine flk2/flt3. It wasshown that the chimeric receptor conferred transformed properties to NIH3T3 cells and sustained long-term proliferation of the Ba/F3 cell line(a murine IL-3-dependent hematopoietic cell line which generates Blymphocytes in vivo) in the absence of IL-3 (Mol. Cell. Biol.,13(10):6572-6585 [1993]). It was shown that flk2/flt3 interacts with thep85 subunit of PI 3'-kinase and induced tyrosine phosphorylation ofPLCγ, GAP and Shc proteins. PI 3'-kinase, PLCγ, GAP and Shc proteins areintracellular substrate proteins which are known to associate with pTKs.

The flk2/flt3 receptor is structurally related to subclass III pTKs suchas α and β platelet-derived growth factor receptors (PDGF-R),colony-stimulating factor (CSF-1, also known as macrophage colonystimulating factor, M-CSF) receptor (c-fms) and Steel factor (also knownas mast cell growth factor, stem cell factor or kit ligand) receptor(c-kit). These receptors form a subfamily of pTKs which have fiveimmunoglobulin-like segments in their ECDs and the intracellularcatalytic domains thereof are interrupted by a specific hydrophilic"interkinase" sequence of variable length. The genes of this pTKsubclass appear to have major growth and/or differentiation functions invarious cells, particularly in the hematopoietic system and in placentaldevelopment (see Rosnet et al. in Genomics, supra). Signaling throughthe c-fms receptor regulates the survival, growth and differentiation ofmonocytes. Steel factor (SLF) which interacts with c-kit stimulates theproliferation of cells in both myeloid and lymphoid lineages and is apotent synergistic factor in combination with other cytokines (Lyman etal., Oncogene, 8:815-822 [1993]).

The flk2/flt3 pTK is mentioned by various other authors. See, forexample, Orlic et al., supra; Birg et al., Blood, 80(10):2584-2593[1992]; and Visser et al., supra.

Lyman et al. refer to the molecular cloning of the transmembrane ligandfor flk2/flt3 which is shown to activate the flk2/flt3 receptor (Cell,75:1157-1167 [1993]). The protein was found to be similar in size andstructure to the cytokines, M-CSF and SLF. The flk2/flt3 ligand wasshown to increase thymidine incorporation in early hematopoietic cellprecursors.

In their earlier publication, Lyman et al. refer to the production ofrabbit polyclonal antibodies against the interkinase domain orC-terminus of flk2/flt3 which immunoprecipitated a major band of 143 kDaand a more diffuse band of 158 kDa. A C-terminal peptide of the flt3sequence containing the final 22 amino acids thereof was used togenerate the antisera. See Lyman et al., Oncogene, 8:815-822 [1993].Maroc et al. also refer to the production of polyclonal antibodiesagainst the C-terminal kinase domain of flk2/flt3 for use in studyingthe biochemical features of this protein (see Oncogene, 8:909-918[1993]). Polyclonal rabbit immune serum was directed against a fusion ofthe interkinase domain of flk2/flt3 with TrpE. However, agonistantibodies which are able to activate the flk2/flt3 receptor haveheretofore not been disclosed.

D. Therapeutic Implications

Chemo- and radiation therapies cause dramatic reductions in blood cellpopulations in cancer patients. At least 500,000 cancer patients undergochemotherapy and radiation therapy in the US and Europe each year andanother 200,000 in Japan. Bone marrow transplantation therapy of valuein aplastic anemia, primary immunodeficiency and acute leukemia(following total body irradiation) is becoming more widely practiced bythe medical community. At least 15,000 Americans have bone marrowtransplants each year. Other diseases can cause a reduction in entire orselected blood cell lineages. Examples of these conditions includeanemia (including macrocytic and aplastic anemia); thrombocytopenia;hypoplasia; immune (autoimmune) thrombocytopenic purpura (ITP); and HIVinduced ITP.

A pharmaceutical product which is able to enhance reconstitution ofblood cell populations in these patients would clearly be of therapeuticbenefit.

Accordingly, it is an object of the present invention to provide agonistantibodies against the flk2/flt3 receptor. The labeled antibodies can beused to detect the flk2/flt3 receptor in biological samples.

It is a further object of this invention to provide a method forenhancing the proliferation or differentiation of primitivehematopoietic cells, thus enhancing repopulation of mature blood celllineages. This is desirable where a mammal has suffered a decrease inhematopoietic or mature blood cells as a consequence of disease,radiation or chemotherapy. This method is also useful for generatingmature blood cell lineages from hematopoietic cells ex vivo.

These and other objects will be apparent to the ordinary artisan uponconsideration of the specification as a whole.

SUMMARY OF THE INVENTION

These objects are accomplished, in one aspect, by providing agonistantibodies against flk2/flt3.

In another aspect, the present invention is based on the observationthat such agonist antibodies against flk2/flk3 are able to enhanceproliferation and differentiation of primitive hematopoietic cells.

Accordingly, the present invention concerns a method for enhancingproliferation or differentiation of primitive hematopoietic cellscomprising contacting the primitive hematopoietic cells with aneffective amount of an agonist antibody against the flk2/flt3 receptor.

In a preferred embodiment, the agonist antibody is a monoclonal antibodydirected against an epitope in the extracellular domain of flk2/flt3.

In a still further aspect, the present invention concerns a method ofenhancing repopulation of blood cell lineages in a mammal comprisingadministering to the mammal a therapeutically effective amount of anagonist antibody against the flk2/flt3 receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the results of the thymidine incorporation assay usingflk2/flt3 receptor transfected into the IL-3 dependent cell line, 32D.In the assay, cells were starved of IL-3 overnight and then stimulatedfor 24 hours and thymidine incorporation was determined. Bothtransfected BAF-3 cells (BAF-3T) and the parent BAF-3 cell line werestimulated with (a) media alone (b) rabbit pre-immune sera (c) 579Apolyclonal antibody or (d) IC2-310 agonist monoclonal antibody.

FIGS. 2A-F show fractionation of fetal liver and bone marrow stem cellpopulations. FIGS. 2A-D show fractionation of AA4⁺ cells from day 14gestation fetal liver. AA4⁺ cells were enriched by immune-panning andwere subsequently stained using Sca-1, CD34, flk-2 and c-kit antibodies.AA4⁺ cells were stained for flk-2 and Sca-1 (FIG. 2A); c-kit and CD34(FIG. 2B); flk-2 and CD34 (FIG. 2C); and flk-2 and c-kit (FIG. 2D).FIGS. 2E & 2F depict fractionation of Lin^(lo) bone marrow progenitorcells with flk-2, CD34 and Sca-1 antibodies. Lin^(lo) bone marrowprogenitor cells were isolated by indirect magnetic bead panning. TheLin cocktail was comprised of RA3-GB2, GR-1, MAC-1, CD4, CD8, Ter-119and CD5. The Lin^(lo) bone marrow cells were stained for flk-2 and CD34(FIG. 2E); and Sca-1 and flk-2 (FIG. 2F), (shown as a dot plot becauseof the very small population of Lin^(lo) Sca⁺ flk-2⁺ cells in themarrow). These experiments were repeated a minimum of four times and thestaining profiles were identical.

FIGS. 3A-D depict dual-parameter fluorescence histograms of AA4⁺ SCA⁺flk-2⁻ (FIG. 3A) and AA4⁺ SCA⁺ flk-2⁺ enriched (FIG. 3B) populationsfollowing cell sorting. FIGS. 3C and 3D illustrate corresponding redfluorescence histograms obtained following acridine orange for AA4⁺ SCA⁺flk-2⁻ (FIG. 3C) and for AA4⁺ SCA⁺ flk-2⁺ enriched populations (FIG.3D). The cursor illustrated the fluorescence intensity utilized todiscriminate G₀ (lower fluorescence intensity) from cycling cells. Thepercentages of cells in each phase of the cell cycle are provided in theinset.

FIG. 4 depicts the effect of IC2-310 on methyl cellulose colonyformation. Hematopoietic cells were seeded into methylcellulose afterbeing cocultivated on the 7-4 fetal liver stromal line for 7 days in thepresence or absence of IC2-310 agonist antibody. The methylcellulosecultures were established under conditions forming either myeloid orlymphoid colonies. Colonies were then stored for CFC after 10 days inculture. Assays were performed in triplicate and repeated in threeseparate experiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

In general, the following words or phrases have the indicated definitionwhen used in the description, examples, and claims:

The term "flk2/flt3" when used herein refers to a polypeptide moleculethat comprises the full-length, native amino acid sequence encoded bythe gene variously known as flk2, flt3 and STK-1, from any species,including the murine and human polypeptides having the amino acidsequences of SEQ ID NO: 2 and SEQ ID NO: 4, respectively, or amino acidsequence variants of such polypeptides. Generally, the DNA encoding suchvariants is capable of hybridizing under stringent conditions with thenative flk2/flt3 DNA sequence. This definition specifically encompassessoluble forms of flk2/flt3, from natural sources (see, e.g., WO94/01576), synthetically produced in vitro or obtained by geneticmanipulation including methods of recombinant DNA technology, as well asvarious chain combinations of such polypeptides. The amino acid sequencevariants preferably share at least about 65% sequence homology, and morepreferably at least about 75% sequence homology with any domain of anative flk2/flt3 amino acid sequence. The definition specifically coversvariously glycosylated and unglycosylated forms of flt2/flt3. Flk2/flt3receptors from non-human or non-murine mammalian (e.g., bovine, equine,porcine, etc.) species can, for example, be obtained by cross-specieshybridization, using probes obtained from the murine or human DNAsequence (SEQ ID NOS: 1 and 3, respectively) as hybridization probes toisolate the cDNA from the mammalian cDNA libraries.

Stringent conditions are those that (1) employ low ionic strength andhigh temperature for washing, for example, 0.015 M NaCl/0.0015 M sodiumcitrate/0.1% NaDodSO₄ at 50° C.; (2) employ during hybridization adenaturing agent such as formamide, for example, 50% (vol/vol) formamidewith 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodiumcitrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl,0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC and 0.1% SDS.

The expression "extracellular domain" or "ECD" when used herein inrelation to the flk2/flt3 receptor refers to any polypeptide sequencethat shares a ligand binding function of the extracellular domain of theflk2/flt3 receptor. Ligand binding function of the extracellular domainrefers to the ability of the polypeptide to bind at least one flk2/flt3ligand (e.g., the ligand disclosed by Lyman et al., in Cell, supra).Accordingly, it is not necessary to include the entire extracellulardomain since smaller segments are commonly found to be adequate forligand binding. The truncated extracellular domain is generally soluble.This term encompasses polypeptide sequences in which the hydrophobictransmembrane sequence (and, optionally, 1-20 amino acids C-terminaland/or N-terminal of the transmembrane domain) of the mature pTK hasbeen deleted. Thus, the soluble extracellular domain-containingpolypeptide can comprise the extracellular domain and the cytoplasmicdomain of the flk2/flt3 receptor. Alternatively, in the preferredembodiment, the polypeptide comprises only the extracellular domain offlk2/flt3. Generally, the ECD will comprise at least amino acid residues1 to 542 of SEQ ID NOS: 2 or 4.

"Antibodies (Abs)" are proteins which exhibit binding specificity to aspecific antigen. Native antibodies are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one end (V_(L)) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and the lightchain variable domain is aligned with the variable domain of the heavychain. Particular amino acid residues are believed to form an interfacebetween the light and heavy chain variable domains (Clothia et al., J.Mol. Biol., 186:651-663 [1985]; Novotny and Haber, Proc. Natl. Acad.Sci, USA, 82:4592-4596 [1985]).

The term "antibody" is used in the broadest sense and specificallycovers single monoclonal antibodies (including agonist and antagonistantibodies), antibody compositions with polyepitopic specificity, aswell as antibody fragments (e.g., Fab, F(ab')₂, and Fv), so long as theyexhibit the desired biological activity.

The term "variable" refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthrough the variable domains of antibodies. It is concentrated in threesegments called complementarity determining regions (CDRs) orhypervariable regions both in the light chain and the heavy chainvariable domains. The more highly conserved portions of the variabledomains are called the framework (FR). The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting aβ-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the β-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen binding site of antibodies (see Kabat, E.A. etal., Sequences of Proteins of Immunological Interest National Instituteof Health, Bethesda, Md. [1987]). The constant domains are not involveddirectly in binding an antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen bindingfragments, called Fab fragments, each with a single antigen bindingsite, and a residual "Fc" fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab')₂ fragment thathas two antigen combining sites and is still capable of cross-linkingantigen.

"Fv" is the minimum antibody fragment which contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen binding site on thesurface of the V_(H) -V_(L) diner. Collectively, the six CDRs conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH₁) of the heavy chain. Fab' fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH₁ domain including one or morecysteines from the antibody hinge region. Fab'-SH is the designationherein for Fab' in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab')₂ antibody fragments originally wereproduced as pairs of Fab' fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The light chains of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called α, delta, epsilon, γ, and μ, respectively.The subunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

The term "monoclonal antibody" as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier "monoclonal"indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler & Milstein, Nature, 256:495 [1975], or may be madeby recombinant DNA methods [see, e.g., U.S. Pat. No. 4,816,567 (Cabillyet al.)].

The monoclonal antibodies herein specifically include "chimeric"antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567(Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 [1984]).

"Humanized" forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab', F(ab')₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibody maycomprise residues which are found neither in the recipient antibody norin the imported CDR or framework sequences. These modifications are madeto further refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin. For further details see:Jones et al., Nature, 321:522-525 [1986]; Reichmann et al., Nature,332:323-329 [1988]; and Presta, Curr. Op. Struct. Biol., 2:593-596[1992]).

By "agonist antibody" is meant an antibody which is able to bind to, andactivate flk2/flt3. For example, the agonist may bind to theextracellular domain of flk2/flt3 and thereby cause dimerization of thisreceptor, resulting in transphosphorylation and activation of theintracellular catalytic kinase domain thereof. Consequently, this mayresult in stimulation of growth and/or differentiation of cellsexpressing the receptor. As disclosed herein, these cells will generallycomprise primitive stem/progenitor hematopoietic cells and thus theagonist antibodies will cause primitive hematopoietic cells todifferentiate and/or proliferate which will generally lead to arepopulation of mature blood cell lineages. The agonist antibodiesherein are preferably against epitopes within the extracellular domainof flk2/flt3. The term "agonist antibody" covers anti-flk2/flt3 agonistmonoclonal antibodies and anti-flk2/flt3 agonist antibody compositionswith polyepitopic specificity. In the preferred embodiment of theinvention, the antibodies are monoclonal antibodies.

In the most preferred embodiment, the monoclonal antibodies have thesame biological characteristics as the monoclonal antibody produced bythe hybridoma cell line deposited under American Type Culture CollectionAccession No. ATCC HB 11,557. By "biological characteristics" is meantthe in vitro and/or in vivo activities of the monoclonal antibody, e.g.,ability to activate the kinase domain of flk2/flt3, ability to stimulatecell growth and/or differentiation of primitive hematopoietic cells andbinding characteristics of the antibody, etc. Accordingly, the antibodypreferably binds to substantially the same epitope as the anti-flk2/flt3monoclonal antibody disclosed herein. Most preferably, the antibody willalso have substantially the same, or greater, antigen binding affinityof the anti-flk2/flt3 monoclonal antibody disclosed herein. To determinewhether a monoclonal antibody has the same specificity as theanti-flk2/flt3 antibody specifically disclosed (i.e., the antibodyhaving the ATCC deposit No. HB 11,557), one can, for example, use acompetitive ELISA binding assay.

An "isolated" polypeptide (e.g., antibody) means polypeptide which hasbeen identified and separated and/or recovered from a component of itsnatural environment. Contaminant components of its natural environmentare materials which would interfere with any diagnostic or therapeuticuse for the polypeptide, and may include enzymes, hormones, and otherproteinaceous or nonproteinaceous solutes. In preferred embodiments, forexample, a polypeptide product comprising a monoclonal antibody of thepresent invention will be purified from a cell culture or othersynthetic environment (1) to greater than 95% by weight of protein asdetermined by the Lowry method, and most preferably more than 99% byweight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a gas- orliquid-phase sequenator (such as commercially available AppliedBiosystems sequenator Model 470, 477, or 473), or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain.

The term "therapeutically effective amount" is used to refer to anamount of any given molecule sufficient for the prevention or treatmentof a specified physiological condition or symptom. The therapeuticallyeffective amount of the agonist antibody to be administered will begoverned by considerations such as the disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners and isthe minimum amount necessary to repopulate mature blood cell lineages inpatients having undergone chemo- or radiation therapy or bone marrowtransplantation therapy or any of the other conditions or diseasesmentioned herein.

By "primitive hematopoietic cells" is meant the most primitive or mostuncommitted blood cells of the hematopoietic system. The blood cells maycomprise totipotent stem cells and/or cells which are slightly committedto a particular blood cell lineage (i.e., multipotent cells).

The term "mammal" refers to any animal classified as a mammal, includinghumans, domestic and farm animals, and zoo, sports, or pet animals, suchas dogs, horses, cats, cows, etc. Preferably, the mammal herein ishuman.

The term "cytokine" is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormone,insulin-like growth factors, human growth hormone, N-methionyl humangrowth hormone, bovine growth hormone, parathyroid hormone, thyroxine,insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH), hematopoietic growth factor, hepaticgrowth factor, fibroblast growth factor, prolactin, placental lactogen,tumor necrosis factor-alpha and -beta, mullerian-inhibiting substance,mouse gonadotropin-associated peptide, inhibin, activin, vascularendothelial growth factor, integrin, thrombopoietin, nerve growthfactors such as NGF-β, platelet-growth factor, transforming growthfactors (TGFs) such as TGF-α and TGF-β, insulin-like growth factor-I and-II, erythropoietin (EPO), osteoinductive factors, interferons such asinterferon-alpha, -beta, and -gamma, colony stimulating factors (CSFs)such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), andgranulocyte-CSF (G-CSF), interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12 and otherpolypeptide factors including LIF, SCF, and kit-ligand. As used hereinthe foregoing terms are meant to include proteins from natural sourcesor from recombinant cell culture. Similarly, the terms are intended toinclude biologically active equivalents; e.g., differing in amino acidsequence by one or more amino acids or in type or extent ofglycosylation.

II. Production of Antibodies

(a) Polyclonal Antibodies

Polyclonal antibodies to flk2/flt3 are generally raised in animals bymultiple subcutaneous (sc) or intraperitoneal (ip) injections of theflk2/flt3 and an adjuvant. It may be useful to conjugate the flk2/flt3or a fragment containing the target amino acid sequence (e.g., the ECDof flk2/flt3) to a protein that is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, where R and R¹ are different alkyl groups.

Animals are immunized against the immunogenic conjugates or derivativesby combining 1 mg or 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freund's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later theanimals are boosted with 1/5 to 1/10 the original amount of conjugate inFreund's complete adjuvant by subcutaneous injection at multiple sites.7 to 14 days later the animals are bled and the serum is assayed forflk2/flt3 antibody titer. Animals are boosted until the titer plateaus.Preferably, the animal is boosted with a conjugate of the same flk2/flt3to a different protein and/or to the same protein through a differentcross-linking reagent. Conjugates also can be made in recombinant cellculture as protein fusions. Also, aggregating agents such as alum areused to enhance the immune response.

(b) Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier"monoclonal" indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the flk2/flt3 monoclonal antibodies of the invention may bemade using the hybridoma method first described by Kohler & Milstein,Nature, 256:495 [1975], or may be made by recombinant DNA methods (U.S.Pat. No. 4,816,567 (Cabilly et al.)).

In the hybridoma method, a mouse or other appropriate host animal, suchas hamster, is immunized as hereinabove described to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the protein used for immunization. Alternatively,lymphocytes may be immunized in vitro. Lymphocytes then are fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell [Goding, Monoclonal Antibodies;Principles and Practice, pp.59-103 (Academic Press, 1986)].

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh level expression of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA. Human myeloma and mouse-human heteromyeloma cell lines alsohave been described for the production of human monoclonal antibodies(Kozbor, J. Immunol., 133:3001 [1984]; Brodeur, et al., MonoclonalAntibody Production Techniques and Applications, pp.51-63, MarcelDekker, Inc., New York, 1987).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against flk2/flt3.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson & Pollard, Anal.Biochem., 107:220 [1980].

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences,(Cabilly et al., supra; Morrison, et al., Proc. Nat. Acad. Sci., 81:6851[1984]), or by covalently joining to the immunoglobulin coding sequenceall or part of the coding sequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for theflk2/flt3 receptor and another antigen-combining site having specificityfor a different antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For diagnostic applications, the antibodies of the invention typicallywill be labeled with a detectable moiety. The detectable moiety can beany one which is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³ H, ¹⁴ C, ³² p, ³⁵ S, or ¹²⁵ I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; radioactive isotopic labels, such as, e.g., ¹²⁵I, ³² P, ¹⁴ C, or ³ H, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter, et al., Nature, 144:945 [1962]; David, et al., Biochemistry,13:1014 [1974]; Pain, et al., J. Immunol. Meth., 40:219 [1981]; andNygren, J. Histochem. and Cytochem., 30:407 [1982].

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard(which may be a flk2/flt3 receptor or an immunologically reactiveportion thereof) to compete with the test sample analyte (flk2/flt3) forbinding with a limited amount of antibody. The amount of flk2/flt3 inthe test sample is inversely proportional to the amount of standard thatbecomes bound to the antibodies. To facilitate determining the amount ofstandard that becomes bound, the antibodies generally are insolubilizedbefore or after the competition, so that the standard and analyte thatare bound to the antibodies may conveniently be separated from thestandard and analyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insoluble threepart complex. David & Greene, U.S. Pat. No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

(c) Humanized and Human Antibodies

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as "import" residues, whichare typically taken from an "import" variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 [1986]; Riechmann et al., Nature,332:323-327 [1988]; Verhoeyen et al., Science, 239:1534-1536 [1988]), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such "humanized" antibodiesare chimeric antibodies (Cabilly et al., supra), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important in order to reduceantigenicity. According to the so called "best-fit" method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 [1993]; Chothia and Lesk, J. Mol. Biol., 196:901[1987]). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 [1992]; Presta et al., J. Immnol., 151:2623 [1993]).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products using threedimensional models of the parental and humanized sequences. Threedimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequence so that thedesired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.For further details see WO 94/04679 published Mar. 3, 1994, thedisclosure of which is incorporated herein by reference.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255[1993]; Jakobovits et al., Nature, 362:255-258 [1993]; Bruggermann etal., Year in Immuno., 7:33 [1993]. Human antibodies can also be producedin phage display libraries (Hoogenboom and Winter, J. Mol. Biol.,227:381 [1991]; Marks et al., J. Mol. Biol., 222:581 [1991]).

The techniques of Cote et al. and Boerner et al. are also available forthe preparation of human monoclonal antibodies (Cote et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 [1985] and Boerner etal., J. Immunol., 147(1):86-95 [1991]).

(d) Screening for Agonist Antibodies

In order to screen for antibodies which are agonists for the flk2/flt3receptor, the tyrosine phosphorylation assay of Holmes et al. (Science,256:1205-1210 [1992]) is available. This assay is described in detail inExample 1C herein. Alternatively, a thymidine incorporation assay usingthe flk2/flt3 receptor transfected into an IL-3 dependent cell line canbe performed. (See Example 1C herein).

III. Therapeutic uses for Anti-flk2/flt3 Agonist Antibodies

The agonist antibodies of the present invention can be used to enhancerepopulation of mature blood cell lineages in cells having undergonechemo- or radiation therapy or bone marrow transplantation therapy.Generally, the antibodies will act via an enhancement of theproliferation and/or differentiation of primitive hematopoietic cells.The antibodies may, for example, enhance the proliferation anddifferentiation of myeloid and lymphoid lineages. The agonist antibodiesare similarly useful for treating diseases characterized by a decreasein blood cells. Examples of these diseases include: anemia (includingmacrocytic and aplastic anemia); thrombocytopenia; hypoplasia; immune(autoimmune) thrombocytopenic purpura (ITP); and HIV induced ITP. Also,the agonist antibodies are useful for treating patients having suffereda hemorrhage.

The antibodies disclosed herein may be administered to a human patient,in a pharmaceutically acceptable dosage form, suitable for intravenous,subcutaneous or intramuscular administration. Such dosage formsencompass pharmaceutically acceptable carriers that are inherentlynontoxic and nontherapeutic. Examples of such carriers include ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts, or electrolytes such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, and polyethyleneglycol. The molecules will typically be formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml.

Pharmaceutical compositions may be prepared and formulated in dosageforms by methods known in the art; for example, see Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15thEdition 1975.

From about 1 to 500 μg/kg, preferably about 1 to 100 μg/kg, morepreferably about 10 to 100 μg/kg, most preferably about 10 to 50 μg/kgof antibody is a suitable initial candidate dosage for administration tothe patient, whether, for example, by one or more separateadministrations, or by continuous infusion. For repeated administrationsover several days or longer, depending on the condition, the treatmentis repeated until a desired suppression of disease symptoms occurs orthe desired improvement in the patient's condition is achieved. The dosemay be readministered at intervals ranging from once a week to onceevery six months. The determination of the optimum dosage and of optimumroute and frequency of administration is well within the knowledge ofthose skilled in the art. Similarly, dosages for other molecules withinthe scope of the present invention can be determined without excessiveexperimentation.

The treatment according to the present invention can be combined withother therapies for enhancing repopulation of hematopoietic blood celllineages. For example, the antibodies can be co-administered with othercytokines or hematopoietic growth factors which are capable of enhancingthe proliferation and/or differentiation of hematopoietic cells (e.g.,Epo, the interleukins; IL-1, IL-3, IL-6, IL-11, GM-CSF, G-CSF, M-CSF,SLF, LIF, TNF, lymphotoxin, flk2/flt3 ligand, kit-ligand, IGF-1 andγ-interferon, etc).

IV. Non-Therapeutic uses for Anti-flt2/flt3 Antibodies

The anti-flk2/flt3 antibodies are useful in diagnostic assays forflt2/flk3, e.g., detecting its expression in specific cells, tissues, orserum. The antibodies are labeled flk2/flt3 and/or are immobilized on aninsoluble matrix. In one embodiment of a receptor binding assay, anantibody composition is immobilized on an insoluble matrix, the testsample is contacted with the immobilized antibody composition to adsorbthe flk2/flt3, and then the immobilized family members are contactedwith a plurality of antibodies specific for each member, each of theantibodies being individually identifiable as specific for apredetermined family member, as by unique labels such as discretefluorophores or the like. By determining the presence and/or amount ofeach unique label, the relative proportion and amount of each familymember can be determined.

The antibodies also are useful for the affinity purification offlk2/flt3 from recombinant cell culture or natural sources. Generalaffinity purification techniques are well known in the art, and any ofthese may be used for this purpose.

Suitable diagnostic assays for the anti-flk2/flt3 antibodies are wellknown per se. For example, competitive, sandwich and steric inhibitionimmunoassay techniques are useful. The competitive and sandwich methodsemploy a phase-separation step as an integral part of the method whilesteric inhibition assays are conducted in a single reaction mixture.Fundamentally, the same procedures are used for the assay of flk2/flt3and for substances that bind flk2/flt3, although certain methods will befavored depending upon the molecular weight of the substance beingassayed. Therefore, the substance to be tested is referred to herein asan analyte, irrespective of its status otherwise as an antigen orantibody, and proteins that bind to the analyte are denominated bindingpartners, whether they be antibodies, cell surface receptors, orantigens.

Analytical methods for flk2/flt3 or its antibodies all use one or moreof the following reagents: labeled analyte analogue, immobilized analyteanalogue, labeled binding partner, immobilized binding partner, andsteric conjugates. The labeled reagents also are known as "tracers."

The label used (and this is also useful to label flk2/flt3 nucleic acidfor use as a probe) is any detectable functionality that does notinterfere with the binding of analyte and its binding partner. Numerouslabels are known for use in immunoassay, examples including moietiesthat may be detected directly, such as fluorochrome, chemiluminscent,and radioactive labels, as well as moieties, such as enzymes, that mustbe reacted or derivatized to be detected. Examples of such labelsinclude the radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luciferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malatedehydrogenase, urease, peroxidase such as horseradish peroxidase (HRP),alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

Those of ordinary skill in the art will know of other suitable labelsthat may be employed in accordance with the present invention. Thebinding of these labels to flk2/flt3, antibodies, or fragments thereofcan be accomplished using standard techniques commonly known to those ofordinary skill in the art. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the polypeptide with theabove-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. No. 3,940,475 (fluorimetry) and U.S. Pat. No.3,645,090 (enzymes); Hunter et al., Nature, 144:945 [1962]; David etal., Biochemistry, 13:1014-1021 [1974]; Pain et al., J. Immunol.Methods, 40:219-230 [1981]; Nygren, J. Histochem. and Cytochem.,30:407-412 [1982]; O'Sullivan et al., Methods in Enzymology, ed. J. J.Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, N.Y.1981), pp. 147-166; Kennedy et al., Clin. Chim. Acta, 70:1-31 [1976];and Schurs et al., Clin. Chim. Acta, 81:1-40 [1977]. Coupling techniquesmentioned in the lattermost reference are the glutaraldehyde method, theperiodate method, the dimaleimide method, and them-maleimidobenzyl-N-hydroxysuccinimide ester method.

In the practice of the present invention, enzyme labels are a preferredembodiment. No single enzyme is ideal for use as a label in everyconceivable assay. Instead, one must determine which enzyme is suitablefor a particular assay system. Criteria important for the choice ofenzymes are turnover number of the pure enzyme (the number of substratemolecules converted to product per enzyme site per unit of time), purityof the enzyme preparation, sensitivity of detection of its product, easeand speed of detection of the enzyme reaction, absence of interferingfactors or of enzyme-like activity in the test fluid, stability of theenzyme and its conjugate, availability and cost of the enzyme and itsconjugate, and the like. Included among the enzymes used as preferredlabels in the assays of the present invention are alkaline phosphatase,HRP, beta-galactosidase, urease, glucose oxidase, glucoamylase, malatedehydrogenase, and glucose-6-phosphate dehydrogenase. Urease is amongthe more preferred enzyme labels, particularly because of chromogenic pHindicators that make its activity readily visible to the naked eye.

Immobilization of reagents is required for certain assay methods.Immobilization entails separating the binding partner from any analytethat remains free in solution. This conventionally is accomplished byeither insolubilizing the binding partner or analyte analogue before theassay procedure, as by adsorption to a water-insoluble matrix or surface(Bennich et al., U.S. Pat. No. 3,720,760), by covalent coupling (forexample, using glutaraldehyde cross-linking), or by insolubilizing thepartner or analogue afterward, e.g., by immunoprecipitation.

Other assay methods, known as competitive or sandwich assays, are wellestablished and widely used in the commercial diagnostics industry.

Competitive assays rely on the ability of a tracer analogue to competewith the test sample analyte for a limited number of binding sites on acommon binding partner. The binding partner generally is insolubilizedbefore or after the competition and then the tracer and analyte bound tothe binding partner are separated from the unbound tracer and analyte.This separation is accomplished by decanting (where the binding partnerwas preinsolubilized) or by centrifuging (where the binding partner wasprecipitated after the competitive reaction). The amount of test sampleanalyte is inversely proportional to the amount of bound tracer asmeasured by the amount of marker substance. Dose-response curves withknown amounts of analyte are prepared and compared with the test resultsto quantitatively determine the amount of analyte present in the testsample. These assays are called ELISA systems when enzymes are used asthe detectable markers.

Another species of competitive assay, called a "homogeneous" assay, doesnot require a phase separation. Here, a conjugate of an enzyme with theanalyte is prepared and used such that when anti-analyte binds to theanalyte the presence of the anti-analyte modifies the enzyme activity.In this case, flk2/flt3 or its immunologically active fragments areconjugated with a bifunctional organic bridge to an enzyme such asperoxidase. Conjugates are selected for use with anti-flk2/flt3 so thatbinding of the anti-flk2/flt3 inhibits or potentiates the enzymeactivity of the label. This method per se is widely practiced under thename of EMIT.

Steric conjugates are used in steric hindrance methods for homogeneousassay. These conjugates are synthesized by covalently linking alow-molecular-weight hapten to a small analyte so that antibody tohapten substantially is unable to bind the conjugate at the same time asanti-analyte. Under this assay procedure the analyte present in the testsample will bind anti-analyte, thereby allowing anti-hapten to bind theconjugate, resulting in a change in the character of the conjugatehapten, e.g., a change in fluorescence when the hapten is a fluorophore.

Sandwich assays particularly are useful for the determination offlk2/flt3 or flk2/flt3 antibodies. In sequential sandwich assays animmobilized binding partner is used to adsorb test sample analyte, thetest sample is removed as by washing, the bound analyte is used toadsorb labeled binding partner, and bound material is then separatedfrom residual tracer. The amount of bound tracer is directlyproportional to test sample analyte. In "simultaneous" sandwich assaysthe test sample is not separated before adding the labeled bindingpartner. A sequential sandwich assay using an anti-flk2/flt3 monoclonalantibody as one antibody and a polyclonal anti-flk2/flt3 antibody as theother is useful in testing samples for flk2/flt3 activity.

The agonist antibodies bind to and activate flk2/flt3 and are thereforeuseful diagnostic tools for further characterizing the biologicalactivity of this receptor in vitro and/or in vivo. The agonistantibodies are also useful for diagnostic purposes. Similarly, theantibodies can be contacted with primitive hematopoietic cells andthereby lead to the proliferation and/or differentiation of mature bloodcell lineages. This is useful where mature blood cells are required forscientific or therapeutic purposes or where the differentiation of cellsis under investigation.

The foregoing are merely exemplary diagnostic assays for flk2/flt3antibodies. Other methods now or hereafter developed for thedetermination of these analytes are included within the scope hereof,including the bioassays described above.

All citations throughout the specification and the references citedtherein are hereby expressly incorporated by reference.

EXAMPLE 1 Production of Agonist Antibodies Against the Flk2/Flt3Receptor

A. Cloning of Murine Flk2/Flt3 Receptor

The murine flk2/flt3 receptor was cloned by RT-PCR from RNA isolatedfrom midgestation mouse fetal livers. Six sets of overlapping primerswere designed to the murine flt-3 sequence disclosed by Rosnet et al.(Oncogene, 6:1641-1650 [1991]). These primers were designed to amplifythree segments of the gene nucleotides 1-1307, 1308-1992, 1993-3096. ThePCR products were then subcloned into pRK5.A. The sequence of thefull-length flk2/flt3 gene was identical to the murine flt-3 genesequence published by Rosnet et al., supra.

A flk2/flt3 extracellular domain (ECD) IgG₁ Fc fusion gene wasconstructed and fusion protein produced as previously described (Bennettet al., J. Biol. Chem., 266:23060-23067 [1991]). The fusion protein waspurified using protein A sepharose and the purified fusion protein wasused for the generation of agonist antibodies against the flk2/flt3receptor ECD.

B. Production of Polyclonal and Monoclonal Antibodies

Polyclonal antibodies were generated in New Zealand White rabbitsagainst the flk2/flt3 fusion protein. 4 μg of fusion protein in 100 μLPBS was emulsified with 100 μL Freund's adjuvant (complete adjuvant forthe primary injection and incomplete adjuvant for all boosts). For theprimary immunization and the first boost, the protein was injecteddirectly into the popliteal lymph nodes (Sigel et al., Methods Enzymol.,93:3-12 [1983]). For subsequent boosts, the protein was injected intosubcutaneous and intramuscular sites. 1.3 μg protein/kg body weight wasinjected every 3 weeks with bleeds taken 1 and 2 weeks following eachboost. Baf-3 cells were transfected with the full length flk2/flt3 geneand used to determine the specificity of antibodies raised to theflk2/flt3-IgG fusion. Significant peak shifts were observed in flk2/flt3expressing clones as compared to either pre-immune serum or vector alonetransfectant controls. The polyclonal antiserum 579A was derived.

Anti-flk2/flt3 monoclonal antibodies were produced by hyperimmunizingBALB/c mice intraperitoneally with the flk2/flt3 extracellular domain(ECD)-human IgG, Fc fusion protein in RIBI adjuvant (RIBI ImmunoChemResearch, Hamilton, Mont.) and fusing Syrian hamster splenocytes withthe mouse myeloma cell line P3X63 Ag8 U.1 (Sanchez-Madrid et al.,Immunol. J., 130:309 [1983]). The antibodies were purified from ascitesfluid using protein A-Sepharose (Repligen Corp., Cambridge, Mass.) andestablished affinity chromatography methods (Goding, J. Immunol.Methods, 20:241-253 [1978]). Flk2/flt3 specificity was assessed by flowcytometry analysis of BAF-3 cells transfected with the full-lengthreceptor. The non-transfected parental line was used as a control. The579A rabbit and polyclonal sera and IC2-514 monoclonal antibody werealso demonstrated to immunoprecipitate the flk2/flt3 receptor from S³⁵-methionine labeled transfected cells lines.

Hamster anti-murine flk2/flt3 hybridomas IC2-514 and IC2-310 resultedfrom the screening. Hybridoma IC2-310 was deposited with the ATCC onMar. 4, 1994 under accession number ATCC HB 11,557.

Agonist polyclonal and monoclonal antibodies were screened for, usingthe techniques disclosed below.

C. Assays for Agonistic Antibodies

Agonistic activity of the 579A polyclonal antisera and IC2-310monoclonal antibody was determined using two assay systems: (a) aphosphotyrosine assay using the full length murine flk2/flt3 receptorexpressed in the IL-3-dependent cell line BAF-3, and (b) a thymidineincorporation assay using the full-length receptor expressed in the IL-3dependent cell line BAF-3. The BAF-3 cells were electroporated with thefull-length flk2/flt3 receptor as previously described (Colosi et al.,J. Biol. Chem., 268:12617-12623 [1992]).

Tyrosine phosphorylation experiments were performed using flk2/flt3receptor transfected into IL-3 dependent cells as previously described(Holmes et al., Science, 256:1205-1210 [1992]). Briefly, transfectedBaf-3 cells (BAF-3T) or non-transfected BAF-3 cells (BAF-3) were starvedof IL-3 and incubated with suspected agonist antibody (hybridomasupernatant polyclonal antisera diluted 1:20) or irrelevant controlantibody for 30 minutes, lysed in IP lysis buffer (1% NP-40 , 1 mM EDTA,200 mM NaCl, 50 mM Tris Hcl pH 8.0, 2 mM PMSF, 2.5 mM Na₃ VO₄) andimmunoprecipitated with 579A polyclonal anti-sera. Immunoprecipitatedlysates were separated on a 4-12% SDS-PAGE gradient gel, transferred tonitrocellulose and Western blotted using the antiphosphotyrosineantibody 4G10. Immunoblotting of the immunoprecipitated material usinganti-phosphotyrosine antibodies demonstrated the phosphorylation of theflk2/flt3 receptor in response to both IC2-310 and 579A, with nophosphorylation of the 160 kD band observed for BAF-3 cells, untreatedcells, or cells treated with an irrelevant control antibody. Themolecular weight of the flk2/flt3 receptor probably reflects extensiveglycosylation of the receptor as was predicted by sequence analysis(Matthews et al., supra). Similar molecular weight values for thefull-length receptor have been obtained in other studies (Lyman et al.,supra and Maroc et al., 1993, supra).

For the thymidine incorporation assay, 32D cells were starved of IL-3for 24 hours and then stimulated with antibody overnight followed by an8 hour pulse of 1 μCi [³ H] thymidine. Thymidine incorporation was thendetermined using a cell harvester. Both 579A and IC2-310 gave asignificant stimulation of thymidine uptake in the transfected 32Dcells. The specificity of these responses was demonstrated by the lackof response to irrelevant antibodies and to hamster IgG (FIG. 1).Following the discovery of IC2-310 agonist activity, the hybridomasupernatant was purified by protein-A affinity. The purified antibodyretained activity at concentrations between 10-40 μg/ml. All subsequentin vitro assays routinely used 40 μg/ml.

The polyclonal antibody (579A) and IC2-310 monoclonal antibody weredemonstrated in each of the above assays to be capable of activating theflk2/flt3 receptor.

EXAMPLE 2 Repopulation of the Hematopoietic System

This example established whether or not the flk2/flt3 receptor wasexpressed on hematopoietic stem cells capable of long-term engraftmentof lethally irradiated hosts.

A. Isolation of Hematopoietic Stem Cell Populations

Hematopoietic stem cell populations were isolated from AA4³⁰ cellsderived from midgestation fetal liver as previously described (Jordan etal., Cell, 61:953-963 [1990]). The AA4⁺ cells were fractionated intoSca⁺ and Sca⁻ sub-populations using the ly6 A/E phycoerythrin conjugate(Pharmingen). The AA4 CD34⁺ or CD34⁻ populations were derived using anaffinity purified rabbit anti-mouse CD34 (Baumhueter et al., Science,262:436-438 [1993]). The purified IC2-514 monoclonal antibody discussedin Example 1 was conjugated to biotin or phycoerythrin for FACSanalysis. The anti-mouse c-kit biotin conjugate was purchased fromPharmingen and all secondary and LIN cocktail antibodies were purchasedfrom Caltag. Bone marrow hematopoietic stem cells were obtained from8-12 week old C.57B/6 ly 5.1 or 5.2 mice. The mononuclear cell fractionwas isolated by density gradient (Accudenz, Accurate Biochemicals) andstained with the LIN cocktail antibodies as previously described (Jordanet al, supra). LIN stained cells were removed via magnetic beaddepletion (Dynal, Inc., Ploemacher et al., Blood, 74:2755-2763 [1989])and the Lin^(lo) population was stained using the appropriateantibodies. Stained cells were selected by propidium iodine (1 μg/ml)exclusion and separated on an Elite flow cytometer (Coulter Electronics,Haileah, Fla.).

B. Competitive Repopulation

The competitive repopulation technique employed in the followingexperiment allows for comparison of the stem cell content of each of thederived populations. Competitive repopulation determines theproliferative capacity of two populations of donor hematopoietic cells(Harrison et al., Hematol. E., 21:206-219 [1993]). In this instance, onemillion bone marrow cells were used as the competitor and the stem cellcontent of the potential stem cell population, for example, AA4⁺ Sca⁺flk-2⁺ was measured relative to the competitor population.

The stem cell equivalents (SCE) per 10,000 cells were determined usingcompetitive repopulation analysis of genetically marked fetal liver orbone marrow in C57BL/6 mice allelic at the Ly 5 locus, designated Ly 5.1and Ly 5.2. Ly 5.1 expression in the Ly 5.2 mice was determined atselected intervals post engraftment.

Stem cell populations were isolated from young adult C57B1/6 Ly 5.1mice. Young adult male C57B/6 Ly 5.2 mice were obtained from NCI andused as recipients. A minimum of five animals was used per experimentalgroup. Whole body irradiation (1100 cGy, 190 cGy/min) was administeredas a single dose from a C137 source. In general, one million bone marrowcells from the 5.2 mice were used as competitors and the stem cellcontent of 1×10⁴ ly 5.1 cells of the potential stem cell population,e.g., AA4⁺ Sca⁺ flk2⁻ was measured relative to the competitorpopulation. Cells were administered via tail vein injection andperipheral blood samples (50-100 μl) were obtained via the retro-orbitalsinus 4 weeks, 12 weeks and 6 months post-reconstitution. The percentageof ly 5.1 donor cells was determined by staining with biotin conjugatedly 5.2 monoclonal (A20.1.7). To confirm repopulation by the donor ly 5.1cells in all lineages, peripheral blood cells and the bone marrowmononuclear fraction were stained with the following antibodies; B220(β-cell lineages), CD4/8 (T cell lineages), Gr-1/Mac-1 (myeloidlineages).

The number of stem cells in each sample (stem cell equivalents, SCE)were determined as described by Kiefer et al., (Blood, 78:2577-2582[1991]). The stem cell content of the bone marrow was estimated at onestem cell per 10⁵ total bone marrow cells (Harrison et al., supra).Therefore, the 1×10⁶ bone marrow cells used as a competitor contained 10stem cells. This means the contribution of the ly 5.1 stem cellpopulation being tested is therefore defined by the equation:

    x÷(10+x)=mean fraction of % ly 5.1 repopulation

    x=SCE

The results are shown in Table 1. All the lineages were reconstituted bydonor 5.1 cells. The results from the AA4⁺ kit⁺ flk-2^(+/-) isolationsrepresent an 8 week time point.

                                      TABLE 1                                     __________________________________________________________________________              4 WEEKS  12 WEEKS 24 WEEKS                                                    % 5.1 SCE                                                                              % 5.1 SCE                                                                              % 5.1 SCE                                         __________________________________________________________________________    AA4.sup.+ 72 ± 3+  Flk-2.sup.+                                                             13 83 ± 4                                                                           24 90 ± 2                                                                           45                                          AA4.sup.+ 68 ± 3+  Flk-2 (.sup.-)                                                          11 89 ± 5                                                                           41 92 ± 2                                                                           57                                          AA4.sup.+  41 ± 12  Flk-2 .sup.+                                                            4  66 ± 10                                                                         10 68 ± 9                                                                           11                                          AA4.sup.+  58 ± 18  Flk-2 (.sup.-)                                                          7 77 ± 9                                                                           17 84 ± 4                                                                           26                                          AA4.sup.+ 61 ± 6+  Flk-2.sup.+                                                             16 71 ± 5                                                                           24 --    --                                          AA4.sup.+ 27 ± 7+  Flk-2 (.sup.-)                                                           4  46 ± 10                                                                          9 --    --                                          AA4.sup.+ 75 ± 4.+  Kit.sup.+                                                              30 84 ± 5                                                                           39 70 ± 3                                                                           23                                          AA4.sup.+ 42D34.sup.+                                                                          7  51 ± 14                                                                         10  49 ± 24                                                                          9                                          AA4.sup.+ 2 ± 2.+  CD34 (.sup.-)                                                            0 23 ± 6                                                                            3  4 ± 3                                                                            0                                          LIN.sup.10 CD34.sup.+  Flk-2.sup.+                                                      51    10  44 ± 25                                                                          8  53 ± 22                                                                         11                                          LIN.sup.10 Sca.sup.+  Flk-2.sup.+                                                       50    10 40 ± 6                                                                            7 25 ± 1                                                                            3                                          LIN.sup.10 Sca.sup.+  Flk-2 (.sup.-)                                                    57    13  53 ± 10                                                                         11  67 ± 12                                                                         20                                          __________________________________________________________________________

In the fetal liver, the AA4.1 antibody (AA4) delineates a population ofone percent of the cells in which all the totipotent stem cell activityresides (Jordan et al., supra). The AA4⁺ population can be furtherenriched using antibodies to the Sca-1 antigen (ly6A/E). The AA4⁺ Sca⁺population contains all the totipotent stem cell activity (Matthews etal., supra). Experiments using the CD34 polyclonal antibody demonstratedthat 60% of the AA4⁺ population are CD34 positive (FIG. 1). Furthermore,repopulation studies showed all the stem cell activity to reside in theAA4⁺ CD34⁺ fraction and that the AA4⁺ CD34⁻ population did notrepopulate (Table 1). It is of further interest to note that all AA4⁺CD34⁺ cells are positive for c-kit expression (FIG. 2) and that all AA4⁺Sca⁺ cells are also CD34 positive. It was also demonstrated that thec-kit positive fraction of the AA4⁺ population contained all stem cellactivity. The AA4⁺ Sca⁺, AA4⁺ CD34⁺, and the AA4⁺ kit⁺ stem cellpopulations were used to investigate stem cell expression of theflk2/flt3 receptor (FIG. 2). Repopulation studies demonstrated thatflk2/flt3 receptor can be expressed on stem cells but that bothflk2/flt3 positive and negative stem cell fractions give rise to longterm reconstitution (Table 1). In experiments using bone marrow as thesource for stem cells, the mononuclear fraction was segregated into aLin^(lo) population by immunomagnetic bead separation using the Lincocktail of antibodies to mature hematopoietic cell types (Ploemacher etal., Blood, 74:2755-2763 [1989]). The Lin^(lo) mononuclear cells werefractionated into Lin^(lo) CD34⁺ and Lin^(lo) Sca⁺ stem cell fractions(FIG. 2). In accordance with the fetal liver populations, these bonemarrow stem cell populations gave both flk2/flt3 positive or flk2/flt3negative sub-populations. Both of is these sub-populations gave rise tolong term repopulation (Table 1).

The above experiments demonstrate the high stem cell content of the AA4⁺CD34⁺ kit⁺ and AA4³⁰ Sca³⁰ flk-2³¹ populations. Furthermore, theyindicate a general trend suggesting that the flk2/flt3 negative cellpopulations have a greater stem cell content when compared to therelevant flk2/flt3 positive population (Table 1). It is noteworthy thatin the marrow there are very few Lin^(lo) Sca⁺ flk-2⁻ cells, most of theLin^(lo) Sca⁺ population being flk2/flt3 positive (FIG. 2). Thefractionation of Lin^(lo) mononuclear cells from the bone marrow intoflk-2⁺ and flk-2⁻ cells demonstrated that the Lin^(lo) flk-2⁺ cells weremore potent at short term repopulation of the irradiated host (3.47versus 1.25 stem cell equivalents per 10,000 cells). Indeed,repopulation from the Lin^(lo) flk-2⁺ cells was minimal after 12 weeks.This suggests that the Lin^(lo) flk-2⁺ population is comprised of morecommitted progenitors than its flk-2⁻ counterpart.

These reconstitution experiments of lethally irradiated mice clearlydemonstrate that the flk2/flt3 receptor tyrosine kinase is expressed onstem cell populations, but quite clearly, not all stem cells expressflk2/flt3. This finding was confirmed in all the different fetal liverand bone marrow stem cell populations isolated. Using competitiverepopulation, it was demonstrated that flk-2 positive stem cellfractions have significantly less repopulating capacity than theirrelative flk-2 negative counterparts.

The most widely used marker for the study of human hematopoietic cellsis cell surface expression of CD34. Various functional assays havedemonstrated that the CD34 positive sub-population from human marrowcontains virtually all primitive hematopoietic cells (Andrews et al.,Med. JE., 16:1721 [1989]; Berenson et al., Invest. JC., 81:951-955[1988]; Civin et al., Immunol. J., 133:157-165 [1984]; and Sutherland etal., Blood, 74:1563-1569 [1989]). The monospecific polyclonal antibodyto murine CD34 clearly demonstrated that in accordance with the humanhomologue, the stem cell activity in murine hematopoiesis is confined tothe CD34⁺ fraction. Therefore, the phenotype of the murine hematopoieticstem cell from the fetal liver is AA4⁺ Lin^(lo) Sca⁺ CD34⁺ kit⁺flk-2^(+/-). From the bone marrow, the phenotype of the stem cell isLin^(lo) Sca⁺ kit⁺ CD34⁺ flk-2^(+/-).

EXAMPLE 3 Cell Cycle Analysis of Flk2/Flt3 Expression

Cell cycle analysis of hematopoietic stem cell populations haspreviously indicated that stem cell populations are heterogeneous inrelation to cell cycle status (Fleming et al., Biol. JC., 122:897-902[1993] and Suda et al., Physiol., JC., 17:308 [1983]). Furthermore,enhanced repopulation has been attributed to those stem cells in the G₀/G₁ phase compared to the actively proliferating S/G₂ /M subset (Fleminget al., supra). If flk2/flt3 expression on stem cells represents apotentially more committed stem cell population, exhibiting decreasedrepopulation capacity, this may be reflected in the cell cycle status.Additionally, it has been suggested that flk2/flt3 RNA expression iscorrelated with cycling stem cells (Orlic et al., supra and Visser etal., supra). Therefore, the cell cycle status of the fetal liver stemcell population AA4⁺ Sca⁺ was determined with respect to flk2/flt3expression (FIG. 3). A two-step acridine orange staining technique wasemployed to differentiate G₀ from G₁ phase cells. When coupled withconventional cell cycle analysis of the DNA content histogram, thismethod allows for the simultaneous analysis of the G₀, G₁, S and G₂ Mcell cycle phase compartments of any cell population.

Two-step acridine orange staining was performed as detailed previously(Darzynkiewicz and Kapuscinski, Flow Cytometry and Sorting, 2nd edition,Wiley-Liss, 291-314; and Baumhueter et al., Science, 262:436-438[1993]). Briefly, cells which had been sorted following dual-parameterimmunofluorescence staining were centrifuged (400×5 min) and resuspendedin RPMI 1640 cell culture medium with 10% fetal bovine serum at a finalconcentration of 10⁶ /ml. To 0.3 ml of this cell suspension, a solutionconsisting of 0.45 ml of 0.1% Triton X-100 in 0.15N NaCl and 0.08N Hclwas added and the mixture incubated for 45 seconds on ice. To thissolution, 1.8 ml of a solution consisting of 12 μm acridine orange(Polysciences, Inc.) in 0.2M Na₂ HPO₄, 0.1M citric acid, 10-³ M Na-EDTAand 0.15M NaCl was added and the sample immediately analyzed by flowcytometry. Red fluorescence (RNA) and green fluorescence (DNA content)were simultaneously collected by the addition of a 560 nm dichroiclong-pass filter coupled with a 525 +/-15 nm bandpass filter (greenfluorescence) and a 630 nm long-pass filter (red fluorescence).

The G₀ sub-population was defined on the basis of the red fluorescenceof peripheral blood mononuclear cells (PBMC) stained in parallel to thepreviously sorted samples. A cursor was placed at the positioncorresponding to the red fluorescence intensity of 97% of PBMC, withcells having higher RNA contents above this position classified ascycling (i.e. G₁, S, G₂ M) populations, and those at or below the cursorclassified as G₀. Enumeration of the proportions of cycling cells wasperformed by conventional cell cycle analysis using the algorithm ofDean and Jett, 1974; available in the Multicycle software (Phoenix FlowSystems, San Diego, Calif.).

It was demonstrated that the major difference between the twosub-populations (AA4⁺ Sca⁺ flk-2⁺ and AA4⁺ Sca⁺ flk- 2⁻) was the greatlyincreased percentage of cells residing in G₀ in the AA4⁺ Sca⁺ flk-2⁻population (FIG. 3). The percentage of cells in S/G₂ /M clearlyindicated that these fetal liver stem cell populations contain manyactively proliferating cells.

Cell cycle analysis of the AA4⁺ kit⁺ stem cell populations demonstrateda much lower percentage of cells in G₀ when compared to the AA4⁺ Sca⁺flk-2⁻ population. However, little difference was found between the AA4⁺kit⁺ flk-2⁺ (G₀ -12%, G₁ -39%, G₂ /M-8%) and the AA4⁺ kit⁺ flk-2⁻ (G₀-7%, G₁ -47%, S-41%, G₂ /M-5%) populations.

These data indicate that the flk2/flt3 receptor is expressed by a subsetof hematopoietic stem cells destined to differentiate to more committedprogenitor cells. This hypothesis gains support from studies that havedemonstrated decreased radioprotective capacity in cycling stem cells(Fleming et al., Biol. JC., 122:897-902 [1993]), and from the expressionof flk2/flt3 mRNA in stem cell fractions believed to be actively cycling(Orlic et al., Blood, 82:762-770 [1993]; and Visser et al., Cells S.,11:49-55 [1993]).

EXAMPLE 4 Hematopoietic Assays of the Flk2/Flt3 Agonist MonoclonalAntibody

A. Dexter Culture Assay System

To assist in the evaluation of the biological function of the IC2-310agonist antibody a Dexter culture assay system was developed usingimmortalized stromal cell lines from the fetal liver.

Fetal liver stromal cells were isolated by infecting primary cultures offetal stroma with the recombinant retrovirus as previously described(Larsson et al., Immunol. D., 1:279-293 [1991]). Viral stocks ofrecombinant retroviruses pZipSvtsA58 were prepared from previouslycharacterized virus producing ψ2 cell lines (Cepko et al., Cell,37:1053-1062 [1984]; and Sharp et al., is Biol. MC., 9:1672-1681 [1989].One of the resultant stromal cell lines designated 7-4 was used in theseexperiments.

The following stromal cell/stem cell co-culture assay was performed.Hematopoietic stem cell populations were seeded at 10⁴ cells/ml on thefetal liver stromal line 7-4 in DMEM/F12 media supplemented with 10%FCS. Co-cultures were incubated at 37° C. for 7 days. The stem cellcontent was determined by competitive repopulation analysis prior to andafter 7 days of coculture. The results obtained from each in vitro assaysystem were confirmed in a minimum of three independent experiments.Growth factors were used at the following concentrations: KL-50 ng/ml;IL-3-1 ng/ml (Genzyme); GM-CSF- 0.2 ng/ml (R & D Systems); PDGF B/B-2ng/ml; and bFGF 2.5 ng/ml (Boehringer Mannheim, USA, Indianapolis,Ind.). Control wells were media alone or media spilled with hamsterIg-G.

After 7 days of coculture the resulting cell populations could onlysustain short-term repopulation of the irradiated host as evidenced bycontribution of donor co-cultivated cells at 4 weeks post-engraftment.However, after this early time point no further contribution from theco-cultivated cells was observed.

Cocultivation upon the 7-4 stroma gave rise to dramatic expansion incell number. See Table 2 below.

                  TABLE 2                                                         ______________________________________                                        CELL                                                                          POPULATION                                                                             CONTROL   310       KL      KL + 310                                 ______________________________________                                        AA4.sup.+  Kit.sup.+                                                                    33 ± 14                                                                             52 ± 2 210 ± 18                                                                           276 ± 12                              Flk-2.sup.+                                                                   AA4.sup.+  Kit.sup.+                                                                   32 ± 3 28 ± 1  71 ± 12                                                                           84 ± 5                                Flk-2 (.sup.-)                                                                AA4.sup.+ SCA.sup.+                                                                       8 ± 0.71                                                                            31 ± 2.1                                                                           120 ± 14                                                                           180 ± 13                              AA4.sup.+  SCA (.sup.-)                                                                   6 ± 0.71                                                                             6 ± 0.84                                                                            13 ± 0.35                                                                          12 ± 0.35                           AA4.sup.+  CD34.sup.+                                                                    12 ± 2.6                                                                             22 ± 2.3                                                                             95 ± 6.1                                                                         129 ± 7                               Kit.sup.+                                                                     AA4.sup.+  CD34.sup.+                                                                  25 ± 5  52 ± 11                                                Flk-2.sup.+                                                                   AA4.sup.+  CD34.sup.+                                                                  12 ± 3 14 ± 5                                                  Flk-2 (.sup.-)                                                                LIN.sup.10 CD34.sup.+                                                                  52 ± 7 173 ± 38                                                Flk-2.sup.+                                                                   ______________________________________                                    

Lineage analysis of the resultant cell populations was performed usingflow cytometric analysis and Wright Geisha staining of cytopsinmaterial. These analyses demonstrated the presence of immatureprogenitor, myeloid and lymphoid cells (see Table 3 below).

                  TABLE 3                                                         ______________________________________                                        CYTOPSIN ANALYSIS                                                                         %        % lg                                                     AA4.sup.+  Sca.sup.+                                                                      Blasts   MNC     % Myeloid                                                                             % Lymphoid                               ______________________________________                                        Media alone 28       47      13      12                                       IC2-310     10       52      30      8                                        IC2-310/KL  15       35      47      3                                        IC2-310/GMCSF                                                                             12       31      52      2                                        IC2-310/KL/GMCSF                                                                           3       23      70      4                                        ______________________________________                                        FACS ANALYSIS                                                                             %        %               %                                        AA4.sup.+  Sca.sup.+                                                                      MAC-1*   Gr-1*   % β220*                                                                          CD4/CD8.sup.+                            ______________________________________                                        Control     65       64      11      36                                       310         76       63      22      46                                       ______________________________________                                    

Many cells maintained the expression of Sca, flk2/flt3, c-kit and CD34.The multilineage potential of this hematopoietic micro-environment wasfurther underscored in the following experiment. Hematopoietic cellswere harvested from the stem cell/stromal cell cocultivation after 7days and stained for FACS analysis using MAC-1 antibody (macrophages),GR-1 (granulocytes), β220 (β cells), CD4/8 (T cell markers). These dataare presented from one representative experiment. The experiments wererepeated a maximum of three times. The results are shown in Table 3.

This was confirmed by cytospin analysis which identified a variety ofhematopoietic cell types including those of the myeloid and lymphoidseries. Cytopsin differentials from the AA4⁺ CD34⁺ kit⁺ stem cellpopulations after cocultivation with stromal cell line 7-4 for 7 dayswere co-cultivated in the presence of the growth factors indicated.Blasts are cells of immature phenotype containing precursors to manylineages. Large mononuclear cells (lg MNC) are cells of intermediatesize and differentiation containing many pre-cursor cells of bothlymphoid and myeloid lineages. Myeloid cells are cells of mature myeloidimages. Lymphold cells are cells displaying lymphocyte or plasma cellcharacteristics. See Table 3.

Stem cells plated in the presence of the IC2-310 agonist antibody gaverise to a greater proliferative event than seen when plated upon 7-4alone. However, IC2-310 did not induce proliferation of the non-stemcell populations such as AA4⁺ Sca⁻. Furthermore, IC2-310 had no effecton the non-flk2/flt3 expressing stem cell populations (Table 2). FACSanalysis of these cells again demonstrated the presence of severalpotential lineages (Table 3). As with cells grown on 7-4 alone, theIC2-310 stimulated cells were only capable of repopulating in the shortterm. The proliferative event enhanced by the IC2-310 antibody wasgreatly increased in combination with KL (Table 2). Cytospin analysis ofthe cocultivated cells demonstrated a significant drop in the percentageof blast cells with a concomitant increase in the percentage of cellsfrom the myeloid lineages including myeloblasts, myelocytes,promyelocytes or metamyelocytes (Table 3). Taken together, these datademonstrate the overall proliferation resulting from stimulation of theflk2/flt3 receptor. Furthermore, they illustrate that in the context ofcocultivation on the 7-4 stromal cell line, this proliferative event isaccompanied by differentiation to more mature hematopoietic phenotypes.

It is clear that activation of the flk2/flt3 receptor promotes theproliferation and differentiation of hematopoietic stem cells when theyare cocultivated with stroma. This proliferation is most clearlyevidenced by the increases in both cell number and colony forming cellsobtained upon activation of stem cells with the IC2-310 agonistantibody. Conversely, the agonist antibody has little effect on non-stemcell populations.

Proliferation of the hematopoietic system by IC2-310 appears to berestricted to stem cell populations and gives rise to an expandedpopulation or more mature hematopoietic phenotypes.

D. Effect of IC2-310 Monoclonal Antibody on Methyl Cellulose ColonyFormation

Hematopoietic colony assays were performed to determine the effects ofthe IC2-310 antibody on the colony forming potential of primitivehematopoietic populations. The methylcellulose assays were performed inthe presence of WEHI conditioned media supplemented with KL in order totest the myeloid potential of the input cell, or alternatively, in thepresence of IL-7 and KL to test the β-lymphoid potential (McNiece etal., Immunol. J., 146:3785-3790 [1991]). Standard myeloidmethylcellulose colony assays were performed using completemethylcellulose medium (Stem Cell Technologies, Inc., #M3430) with theaddition of 50 ng/ml kit ligand, KL (R & D Systems, Minneapolis, Minn.).Colonies were counted after 10 days in culture; only colonies of greaterthan 50 cells were scored. Lymphoid colonies were produced using basemethylcellulose (Stem Cell Technologies) with 50 ng/ml kit ligand and 50ng/ml murine IL-7 (R & D Systems, Minneapolis, Minn.), see McNiece etal., supra. Cytospin analysis of the resultant colonies was performed aspreviously described (Testa and Molineux, Hematopoiesis: Oxford IRLPress).

Hematopoietic stem cell populations plated onto 7-4 cells and thenremoved after 7 days were capable of forming both myeloid colony formingcells (CFC) and lymphoid CFC. When the cocultivation on 7-4 wasperformed in the presence of IC2-310 agonist antibody there was anapproximate 5 fold increase in myeloid CFC and a 12 fold increase inlymphoid CFC (FIG. 4). Cytospin data revealed the myeloid colonies to beof mixed lineage but principally they represented thegranulocyte/macrophage subset. Analysis of the colonies produced in thepresence of IL-7 and KL demonstrated a B220⁺ IgM⁻ phenotype. Most ofthese cells also stained for the S7 marker which is considered to stainβ lineage cells before the pre-β stage (Hardy et al., J. Exp. Med.,173:1213-1225 [1991]). Once again, the proliferative effect of IC2-310was restricted to the stem cell population. No effect was seen on theAA4⁺ Sca⁻ cell population which does not contain stem cells (FIG. 4).

These results support the observations that the IC2-310 antibody is onlycapable of stimulating proliferation of the most primitive hematopoieticcell populations.

Deposit of Materials

The following culture has been deposited with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md., USA (ATCC):

    ______________________________________                                        Hybridoma     ATCC No.    Deposit Date                                        ______________________________________                                        Anti-FLK2/FLT3                                                                              ATCC HB 11,557                                                                            March 4, 1994                                       ______________________________________                                    

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture for 30 years fromthe date of deposit. The organism will be made available by ATCC underthe terms of the Budapest Treaty, and subject to an agreement betweenGenentech, Inc. and ATCC, which assures permanent and unrestrictedavailability of the progeny of the culture to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 USC§122 and the Commissioner's rules pursuant thereto (including 37 CFR§1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if the cultureon deposit should die or be lost or destroyed when cultivated undersuitable conditions, it will be promptly replaced on notification with aviable specimen of the same culture. Availability of the depositedstrain is not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the culture deposited, sincethe deposited embodiment is intended as a single illustration of oneaspect of the invention and any culture that are functionally equivalentare within the scope of this invention. The deposit of material hereindoes not constitute an admission that the written description hereincontained is inadequate to enable the practice of any aspect of theinvention, including the best mode thereof, nor is it to be construed aslimiting the scope of the claims to the specific illustration that itrepresents. Indeed, various modifications of the invention in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description and fall within thescope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 4                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 3521 bases                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #              50GGGGCG CGCCGCTGGG ACCGCATCAC AGGCTGGGCC                      #             100CGCGCG CTCCGGAGGC CATGCGGGCG TTGGCGCAGC                      #             150CTGCTG CTGCTTGTTG TTTTGTCAGT AATGATTCTT                      #             200CCAAGA CCTGCCTGTG ATCAAGTGTG TTTTAATCAG                      #             250GCTCAT CAGCGGGAAA GCCATCATCG TACCGAATGG                      #             300GAAGAC CTCCAGTGTA CCCCGAGGCG CCAGAGTGAA                      #             350AGCGGC CACCGTGGAG GTGGCCGAGT CTGGGTCCAT                      #             400AGCTCG CCACCCCAGG GGACCTTTCC TGCCTCTGGG                      #             450TCCCTG GGCTGCCAGC CGCACTTTGA TTTACAAAAC                      #             500CATGGC CATCTTGAAC GTGACAGAGA CCCAGGCAGG                      #             550ATATTC AGAGCGAAGC CGCCAACTAC ACAGTACTGT                      #             600AGAGAT ACACAGCTGT ATGTGCTAAG GAGACCTTAC                      #             650AAACCA GGATGCACTG CTCTGCATCT CCGAGGGTGT                      #             700TGGAGT GGGTGCTCTG CAGCTCCCAC AGGGAAAGCT                      #             750CCTGCT GTTGTCAGAA AGGAGGAAAA GGTACTTCAT                      #             800AGACAT CAGATGCTGT GCTAGAAATG CACTGGGCCG                      #             850TGTTCA CCATAGATCT AAACCAGGCT CCTCAGAGCA                      #             900TTCCTG AAAGTGGGGG AACCCTTGTG GATCAGGTGT                      #             950GAACCA TGGATTCGGG CTCACCTGGG AGCTGGAAGA                      #            1000AGGGCA GCTACTTTGA GATGAGTACC TACTCCACAA                      #            1050CGGATT CTCTTGGCCT TTGTGTCTTC CGTGGGAAGG                      #            1100TTACAC CTGCTCTTCC TCAAAGCACC CCAGCCAGTC                      #            1150TCCTAG AAAAAGGGTT TATAAACGCT ACCAGCTCGC                      #            1200ATTGAC CCGTACGAAA AGTTCTGCTT CTCAGTCAGG                      #            1250ACGAAT CCGATGCACG TGGATCTTCT CTCAAGCCTC                      #            1300AGAGAG GCCTGGAGGA TGGGTACAGC ATATCTAAAT                      #            1350AACAAG CCAGGAGAGT ACATATTCTA TGCAGAAAAT                      #            1400CACCAA AATGTTCACG CTGAATATAA GAAAGAAACC                      #            1450ATGCCT CAGCCAGCCA GGCGTCCTGT TCCTCTGATG                      #            1500TCTTGG ACCTGGAAGA AGTGTTCGGA CAAATCTCCC                      #            1550AATCCC AGAAGGAGTT TGGAATAAAA AGGCTAACAG                      #            1600AGTGGG TGTCGAGCAG TACTCTAAAT ATGAGTGAGG                      #            1650CTGGTC AAATGCTGTG CGTACAATTC TATGGGCACG                      #            1700CTTTTT AAACTCACCA GGCCCCTTCC CTTTCATCCA                      #            1750TCTATG CGACCATTGG GCTCTGTCTC CCCTTCATTG                      #            1800TTGATC TGCCACAAAT ACAAAAAGCA ATTTAGGTAC                      #            1850GATGAT CCAGGTGACT GGCCCCCTGG ATAACGAGTA                      #            1900TCAGGG ACTATGAATA TGACCTTAAG TGGGAGTTCC                      #            1950GAGTTT GGGAAGGTCC TGGGGTCTGG CGCTTTCGGG                      #            2000CACGGC CTATGGCATT AGTAAAACGG GAGTCTCAAT                      #            2050AGATGC TAAAAGAGAA AGCTGACAGC TGTGAAAAAG                      #            2100GAGCTC AAAATGATGA CCCACCTGGG ACACCATGAC                      #            2150GCTGGG GGCATGCACA CTGTCAGGGC CAGTGTACTT                      #            2200GTTGCT ATGGTGACCT CCTCAACTAC CTAAGAAGTA                      #            2250CACAGG ACATGGACAG AGATTTTTAA GGAACATAAT                      #            2300TACTTT CCAGGCACAT TCAAATTCCA GCATGCCTGG                      #            2350AGTTAC ACCCGCCCTT GGATCAGCTC TCAGGGTTCA                      #            2400CATTCT GAAGATGAGA TTGAATATGA AAACCAGAAG                      #            2450AGAGGA GGAAGATTTG AACGTGCTGA CGTTTGAAGA                      #            2500CGTACC AAGTGGCCAA AGGCATGGAA TTCCTGGAGT                      #            2550CACAGA GACCTGGCAG CCAGGAATGT GTTGGTCACC                      #            2600GAAGAT CTGTGACTTT GGACTGGCCC GAGACATCCT                      #            2650ACGTCG TCAGGGGCAA CGCACGGCTG CCGGTGAAGT                      #            2700AGCTTA TTTGAAGGGA TCTACACAAT CAAGAGTGAC                      #            2750CATCCT TCTCTGGGAG ATATTTTCAC TGGGTGTGAA                      #            2800TTCCTG TCGACGCTAA CTTCTATAAA CTGATTCAGA                      #            2850GAGCAG CCATTCTATG CCACAGAAGG GATATACTTT                      #            2900CTGGGC TTTTGACTCA AGGAAGCGGC CATCCTTCCC                      #            2950TTTTAG GATGTCAGCT GGCAGAGGCA GAAGAAGCGA                      #            3000GGTGGC AACGTCCCAG AACATCCATC CATCTACCAA                      #            3050CAGCAG AGAGGCGGGC TCAGAGCCGC CATCGCCACA                      #            3100TTCACA GAGAAAGAAG TTAGCGAGGA GGCCTTGGAC                      #            3150AGGCTG TAGACCGCAG AGCCAAGATT AGCCTCGCCT                      #            3200TACAGG CCGTTGCTTC GCTGGACTTT TCTCTAGATG                      #            3250TCCAAA GTGACTTCTA TAAAATCAAA CCTCTCCTCG                      #            3300CCAATA ATGAGACTTG TTGGTGAGCC CGCCTACCCT                      #            3350GGCCCC CCAGGCTTGA GGGGAAAGCC ATGTATCTGA                      #            3400TTGTAA ATACGTGAAA CAAACCAAAC CCGTTTTTTG                      #            3450AATATG ATTTTTAAAA ATCTATGTTT TAAAATACTA                      #            3500CTATTT AGTGATATAT TTTATGGATG GAAATAAACT                      #                3521AA A                                                     - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1000 amino                                                        (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Arg Ala Leu Ala Gln Arg Ser Asp Arg Ar - #g Leu Leu Leu Leu             #                 15                                                          - Val Val Leu Ser Val Met Ile Leu Glu Thr Va - #l Thr Asn Gln Asp             #                 30                                                          - Leu Pro Val Ile Lys Cys Val Leu Ile Ser Hi - #s Glu Asn Asn Gly             #                 45                                                          - Ser Ser Ala Gly Lys Pro Ser Ser Tyr Arg Me - #t Val Arg Gly Ser             #                 60                                                          - Pro Glu Asp Leu Gln Cys Thr Pro Arg Arg Gl - #n Ser Glu Gly Thr             #                 75                                                          - Val Tyr Glu Ala Ala Thr Val Glu Val Ala Gl - #u Ser Gly Ser Ile             #                 90                                                          - Thr Leu Gln Val Gln Leu Ala Thr Pro Gly As - #p Leu Ser Cys Leu             #                105                                                          - Trp Val Phe Lys His Ser Ser Leu Gly Cys Gl - #n Pro His Phe Asp             #               120                                                           - Leu Gln Asn Arg Gly Ile Val Ser Met Ala Il - #e Leu Asn Val Thr             #               135                                                           - Glu Thr Gln Ala Gly Glu Tyr Leu Leu His Il - #e Gln Ser Glu Ala             #               150                                                           - Ala Asn Tyr Thr Val Leu Phe Thr Val Asn Va - #l Arg Asp Thr Gln             #               165                                                           - Leu Tyr Val Leu Arg Arg Pro Tyr Phe Arg Ly - #s Met Glu Asn Gln             #               180                                                           - Asp Ala Leu Leu Cys Ile Ser Glu Gly Val Pr - #o Glu Pro Thr Val             #               195                                                           - Glu Trp Val Leu Cys Ser Ser His Arg Glu Se - #r Cys Lys Glu Glu             #               210                                                           - Gly Pro Ala Val Val Arg Lys Glu Glu Lys Va - #l Leu His Glu Leu             #               225                                                           - Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg As - #n Ala Leu Gly Arg             #               240                                                           - Glu Ser Thr Lys Leu Phe Thr Ile Asp Leu As - #n Gln Ala Pro Gln             #               255                                                           - Ser Thr Leu Pro Gln Leu Phe Leu Lys Val Gl - #y Glu Pro Leu Trp             #               270                                                           - Ile Arg Cys Lys Ala Ile His Val Asn His Gl - #y Phe Gly Leu Thr             #               285                                                           - Trp Glu Leu Glu Asp Lys Ala Leu Glu Glu Gl - #y Ser Tyr Phe Glu             #               300                                                           - Met Ser Thr Tyr Ser Thr Asn Arg Thr Met Il - #e Arg Ile Leu Leu             #               315                                                           - Ala Phe Val Ser Ser Val Gly Arg Asn Asp Th - #r Gly Tyr Tyr Thr             #               330                                                           - Cys Ser Ser Ser Lys His Pro Ser Gln Ser Al - #a Leu Val Thr Ile             #               345                                                           - Leu Glu Lys Gly Phe Ile Asn Ala Thr Ser Se - #r Gln Glu Glu Tyr             #               360                                                           - Glu Ile Asp Pro Tyr Glu Lys Phe Cys Phe Se - #r Val Arg Phe Lys             #               375                                                           - Ala Tyr Pro Arg Ile Arg Cys Thr Trp Ile Ph - #e Ser Gln Ala Ser             #               390                                                           - Phe Pro Cys Glu Gln Arg Gly Leu Glu Asp Gl - #y Tyr Ser Ile Ser             #               405                                                           - Lys Phe Cys Asp His Lys Asn Lys Pro Gly Gl - #u Tyr Ile Phe Tyr             #               420                                                           - Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Me - #t Phe Thr Leu Asn             #               435                                                           - Ile Arg Lys Lys Pro Gln Val Leu Ala Asn Al - #a Ser Ala Ser Gln             #               450                                                           - Ala Ser Cys Ser Ser Asp Gly Tyr Pro Leu Pr - #o Ser Trp Thr Trp             #               465                                                           - Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Th - #r Glu Glu Ile Pro             #               480                                                           - Glu Gly Val Trp Asn Lys Lys Ala Asn Arg Ly - #s Val Phe Gly Gln             #               495                                                           - Trp Val Ser Ser Ser Thr Leu Asn Met Ser Gl - #u Ala Gly Lys Gly             #               510                                                           - Leu Leu Val Lys Cys Cys Ala Tyr Asn Ser Me - #t Gly Thr Ser Cys             #               525                                                           - Glu Thr Ile Phe Leu Asn Ser Pro Gly Pro Ph - #e Pro Phe Ile Gln             #               540                                                           - Asp Asn Ile Ser Phe Tyr Ala Thr Ile Gly Le - #u Cys Leu Pro Phe             #               555                                                           - Ile Val Val Leu Ile Val Leu Ile Cys His Ly - #s Tyr Lys Lys Gln             #               570                                                           - Phe Arg Tyr Glu Ser Gln Leu Gln Met Ile Gl - #n Val Thr Gly Pro             #               585                                                           - Leu Asp Asn Glu Tyr Phe Tyr Val Asp Phe Ar - #g Asp Tyr Glu Tyr             #               600                                                           - Asp Leu Lys Trp Glu Phe Pro Arg Glu Asn Le - #u Glu Phe Gly Lys             #               615                                                           - Val Leu Gly Ser Gly Ala Phe Gly Arg Val Me - #t Asn Ala Thr Ala             #               630                                                           - Tyr Gly Ile Ser Lys Thr Gly Val Ser Ile Gl - #n Val Ala Val Lys             #               645                                                           - Met Leu Lys Glu Lys Ala Asp Ser Cys Glu Ly - #s Glu Ala Leu Met             #               660                                                           - Ser Glu Leu Lys Met Met Thr His Leu Gly Hi - #s His Asp Asn Ile             #               675                                                           - Val Asn Leu Leu Gly Ala Cys Thr Leu Ser Gl - #y Pro Val Tyr Leu             #               690                                                           - Ile Phe Glu Tyr Cys Cys Tyr Gly Asp Leu Le - #u Asn Tyr Leu Arg             #               705                                                           - Ser Lys Arg Glu Lys Phe His Arg Thr Trp Th - #r Glu Ile Phe Lys             #               720                                                           - Glu His Asn Phe Ser Phe Tyr Pro Thr Phe Gl - #n Ala His Ser Asn             #               735                                                           - Ser Ser Met Pro Gly Ser Arg Glu Val Gln Le - #u His Pro Pro Leu             #               750                                                           - Asp Gln Leu Ser Gly Phe Asn Gly Asn Ser Il - #e His Ser Glu Asp             #               765                                                           - Glu Ile Glu Tyr Glu Asn Gln Lys Arg Leu Al - #a Glu Glu Glu Glu             #               780                                                           - Glu Asp Leu Asn Val Leu Thr Phe Glu Asp Le - #u Leu Cys Phe Ala             #               795                                                           - Tyr Gln Val Ala Lys Gly Met Glu Phe Leu Gl - #u Phe Lys Ser Cys             #               810                                                           - Val His Arg Asp Leu Ala Ala Arg Asn Val Le - #u Val Thr His Gly             #               825                                                           - Lys Val Val Lys Ile Cys Asp Phe Gly Leu Al - #a Arg Asp Ile Leu             #               840                                                           - Ser Asp Ser Ser Tyr Val Val Arg Gly Asn Al - #a Arg Leu Pro Val             #               855                                                           - Lys Trp Met Ala Pro Glu Ser Leu Phe Glu Gl - #y Ile Tyr Thr Ile             #               870                                                           - Lys Ser Asp Val Trp Ser Tyr Gly Ile Leu Le - #u Trp Glu Ile Phe             #               885                                                           - Ser Leu Gly Val Asn Pro Tyr Pro Gly Ile Pr - #o Val Asp Ala Asn             #               900                                                           - Phe Tyr Lys Leu Ile Gln Ser Gly Phe Lys Me - #t Glu Gln Pro Phe             #               915                                                           - Tyr Ala Thr Glu Gly Ile Tyr Phe Val Met Gl - #n Ser Cys Trp Ala             #               930                                                           - Phe Asp Ser Arg Lys Arg Pro Ser Phe Pro As - #n Leu Thr Ser Phe             #               945                                                           - Leu Gly Cys Gln Leu Ala Glu Ala Glu Glu Al - #a Met Tyr Gln Asn             #               960                                                           - Met Gly Gly Asn Val Pro Glu His Pro Ser Il - #e Tyr Gln Asn Arg             #               975                                                           - Arg Pro Leu Ser Arg Glu Ala Gly Ser Glu Pr - #o Pro Ser Pro Gln             #               990                                                           - Ala Gln Val Lys Ile His Arg Glu Arg Ser                                     #              1000                                                           - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 3475 bases                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #              50AGGGCT GGGCCGGCGC CCTGGGGGAC CCCGGGCTCC                      #             100CGTTGG CGCGCGACGC GGGCACCGTG CCGCTGCTCG                      #             150ATGATA TTTGGGACTA TTACAAATCA AGATCTGCCT                      #             200TTTAAT CAATCATAAG AACAATGATT CATCAGTGGG                      #             250ATCCCA TGGTATCAGA ATCCCCGGAA GACCTCGGGT                      #             300CAGAGC TCAGGGACAG TGTACGAAGC TGCCGCTGTG                      #             350TGCTTC CATCACACTG CAAGTGCTGG TCGATGCCCC                      #             400GTCTCT GGGTCTTTAA GCACAGCTCC CTGAATTGCC                      #             450TTACAA AACAGAGGAG TTGTTTCCAT GGTCATTTTG                      #             500CCAAGC TGGAGAATAC CTACTTTTTA TTCAGAGTGA                      #             550CAATAT TGTTTACAGT GAGTATAAGA AATACCCTGC                      #             600AGACCT TACTTTAGAA AAATGGAAAA CCAGGACGCC                      #             650TGAGAG CGTTCCAGAG CCGATCGTGG AATGGGTGCT                      #             700GGGAAA GCTGTAAAGA AGAAAGTCCA GCTGTTGTTA                      #             750GTGCTT CATGAATTAT TTGGGACGGA CATAAGGTGC                      #             800ACTGGG CAGGGAATGC ACCAGGCTGT TCACAATAGA                      #             850CTCAGA CCACATTGCC ACAATTATTT CTTAAAGTAG                      #             900ATAAGG TGCAAAGCTG TTCATGTGAA CCATGGATTC                      #             950ATTAGA AAACAAAGCA CTCGAGGAGG GCAACTACTT                      #            1000ATTCAA CAAACAGAAC TATGATACGG ATTCTGTTTG                      #            1050GTGGCA AGAAACGACA CCGGATACTA CACTTGTTCC                      #            1100CAGTCA ATCAGCTTTG GTTACCATCG TAGGAAAGGG                      #            1150CCAATT CAAGTGAAGA TTATGAAATT GACCAATATG                      #            1200TCTGTC AGGTTTAAAG CCTACCCACA AATCAGATGT                      #            1250TCGAAA ATCATTTCCT TGTGAGCAAA AGGGTCTTGA                      #            1300TATCCA AGTTTTGCAA TCATAAGCAC CAGCCAGGAG                      #            1350GCAGAA AATGATGATG CCCAATTTAC CAAAATGTTC                      #            1400AAGGAA ACCTCAAGTG CTCGCAGAAG CATCGGCAAG                      #            1450TCTCGG ATGGATACCC ATTACCATCT TGGACCTGGA                      #            1500AAGTCT CCCAACTGCA CAGAAGAGAT CACAGAAGGA                      #            1550GGCTAA CAGAAAAGTG TTTGGACAGT GGGTGTCGAG                      #            1600TGAGTG AAGCCATAAA AGGGTTCCTG GTCAAGTGCT                      #            1650CTTGGC ACATCTTGTG AGACGATCCT TTTAAACTCT                      #            1700TTTCAT CCAAGACAAC ATCTCATTCT ATGCAACAAT                      #            1750TCTTCA TTGTCGTTTT AACCCTGCTA ATTTGTCACA                      #            1800TTTAGG TATGAAAGCC AGCTACAGAT GGTACAGGTG                      #            1850TAATGA GTACTTCTAC GTTGATTTCA GAGAATATGA                      #            1900GGGAGT TTCCAAGAGA AAATTTAGAG TTTGGGAAGG                      #            1950GCTTTT GGAAAAGTGA TGAACGCAAC AGCTTATGGA                      #            2000AGTCTC AATCCAGGTT GCCGTCAAAA TGCTGAAAGA                      #            2050CTGAAA GAGAGGCACT CATGTCAGAA CTCAAGATGA                      #            2100AGCCAC GAGAATATTG TGAACCTGCT GGGGGCGTGC                      #            2150AATTTA CTTGATTTTT GAATACTGTT GCTATGGTGA                      #            2200TAAGAA GTAAAAGAGA AAAATTTCAC AGGACTTGGA                      #            2250GAACAC AATTTCAGTT TTTACCCCAC TTTCCAATCA                      #            2300CATGCC TGGTTCAAGA GAAGTTCAGA TACACCCGGA                      #            2350CAGGGC TTCATGGGAA TTCATTTCAC TCTGAAGATG                      #            2400AACCAA AAAAGGCTGG AAGAAGAGGA GGACTTGAAT                      #            2450AGATCT TCTTTGCTTT GCATATCAAG TTGCCAAAGG                      #            2500AATTTA AGTCGTGTGT TCACAGAGAC CTGGCCGCCA                      #            2550ACCCAC GGGAAAGTGG TGAAGATATG TGACTTTGGA                      #            2600CATGAG TGATTCCAAC TATGTTGTCA GGGGCAATGC                      #            2650AATGGA TGGCCCCCGA AAGCCTGTTT GAAGGCATCT                      #            2700GATGTC TGGTCATATG GAATATTACT GTGGGAAATC                      #            2750GAATCC TTACCCTGGC ATTCCGGTTG ATGCTAACTT                      #            2800AAAATG GATTTAAAAT GGATCAGCCA TTTTATGCTA                      #            2850ATTATA ATGCAATCCT GCTGGGCTTT TGACTCAAGG                      #            2900CCCTAA TTTGACTTCG TTTTTAGGAT GTCAGCTGGC                      #            2950CGATGT ATCAGAATGT GGATGGCCGT GTTTCGGAAT                      #            3000CAAAAC AGGCGACCTT TCAGCAGAGA GATGGATTTG                      #            3050GCAGGC TCAGGTCGAA GATTCGTAGA GGAACAATTT                      #            3100CATCCC TCCACCTATC CCTAACAGGC TGTAGATTAC                      #            3150TTTCAT CACTAAAAGA AAATCTATTA TCAACTGCTG                      #            3200TCTCTA GAAGCCGTCT GCGTTTACTC TTGTTTTCAA                      #            3250AATCAA ATCATCCTGT CACAAGGCAG GAGGAGCTGA                      #            3300GGAGCA TTGATCTGCA TCCAAGGCCT TCTCAGGCCG                      #            3350TGTACC TGAAGTACAG TATATTCTTG TAAATACATA                      #            3400TGCTAA GGAGAAGCTA ATATGATTTT TTAAGTCTAT                      #            3450TGTAAA TTTTTCAGCT ATTTAGTGAT ATATTTTATG                      #             3475 CTAC TACAG                                                 - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 993 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Pro Ala Leu Ala Arg Asp Gly Gly Gln Le - #u Pro Leu Leu Val             #                 15                                                          - Val Phe Ser Ala Met Ile Phe Gly Thr Ile Th - #r Asn Gln Asp Leu             #                 30                                                          - Pro Val Ile Lys Cys Val Leu Ile Asn His Ly - #s Asn Asn Asp Ser             #                 45                                                          - Ser Val Gly Lys Ser Ser Ser Tyr Pro Met Va - #l Ser Glu Ser Pro             #                 60                                                          - Glu Asp Leu Gly Cys Ala Leu Arg Pro Gln Se - #r Ser Gly Thr Val             #                 75                                                          - Tyr Glu Ala Ala Ala Val Glu Val Asp Val Se - #r Ala Ser Ile Thr             #                 90                                                          - Leu Gln Val Leu Val Asp Ala Pro Gly Asn Il - #e Ser Cys Leu Trp             #                105                                                          - Val Phe Lys His Ser Ser Leu Asn Cys Gln Pr - #o His Phe Asp Leu             #               120                                                           - Gln Asn Arg Gly Val Val Ser Met Val Ile Le - #u Lys Met Thr Glu             #               135                                                           - Thr Gln Ala Gly Glu Tyr Leu Leu Phe Ile Gl - #n Ser Glu Ala Thr             #               150                                                           - Asn Tyr Thr Ile Leu Phe Thr Val Ser Ile Ar - #g Asn Thr Leu Leu             #               165                                                           - Tyr Thr Leu Arg Arg Pro Tyr Phe Arg Lys Me - #t Glu Asn Gln Asp             #               180                                                           - Ala Leu Val Cys Ile Ser Glu Ser Val Pro Gl - #u Pro Ile Val Glu             #               195                                                           - Trp Val Leu Cys Asp Ser Gln Gly Glu Ser Cy - #s Lys Glu Glu Ser             #               210                                                           - Pro Ala Val Val Lys Lys Glu Glu Lys Val Le - #u His Glu Leu Phe             #               225                                                           - Gly Met Asp Ile Arg Cys Cys Ala Arg Asn Gl - #u Leu Gly Arg Glu             #               240                                                           - Cys Thr Arg Leu Phe Thr Ile Asp Leu Asn Gl - #n Thr Pro Gln Thr             #               255                                                           - Thr Leu Pro Gln Leu Phe Leu Lys Val Gly Gl - #u Pro Leu Trp Ile             #               270                                                           - Arg Cys Lys Ala Val His Val Asn His Gly Ph - #e Gly Leu Thr Trp             #               285                                                           - Glu Leu Glu Asn Lys Ala Leu Glu Glu Gly As - #n Tyr Phe Glu Met             #               300                                                           - Ser Thr Tyr Ser Thr Asn Arg Thr Met Ile Ar - #g Ile Leu Phe Ala             #               315                                                           - Phe Val Ser Ser Val Ala Arg Asn Asp Thr Gl - #y Tyr Tyr Thr Cys             #               330                                                           - Ser Ser Ser Lys His Pro Ser Gln Ser Ala Le - #u Val Thr Ile Val             #               345                                                           - Glu Lys Gly Phe Ile Asn Ala Thr Asn Ser Se - #r Glu Asp Tyr Glu             #               360                                                           - Ile Asp Gln Tyr Glu Glu Phe Cys Phe Ser Va - #l Arg Phe Lys Ala             #               375                                                           - Tyr Pro Gln Ile Arg Cys Thr Trp Thr Phe Se - #r Arg Lys Ser Phe             #               390                                                           - Pro Cys Glu Gln Lys Gly Leu Asp Asn Gly Ty - #r Ser Ile Ser Lys             #               405                                                           - Phe Cys Asn His Lys His Gln Pro Gly Glu Ty - #r Ile Phe His Ala             #               420                                                           - Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Ph - #e Thr Leu Asn Ile             #               435                                                           - Arg Arg Lys Pro Gln Val Leu Ala Glu Ala Se - #r Ala Ser Gln Ala             #               450                                                           - Ser Cys Phe Ser Asp Gly Tyr Pro Leu Pro Se - #r Trp Thr Trp Lys             #               465                                                           - Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Gl - #u Glu Ile Thr Glu             #               480                                                           - Gly Val Trp Asn Arg Lys Ala Asn Arg Lys Va - #l Phe Gly Gln Trp             #               495                                                           - Val Ser Ser Ser Thr Leu Asn Met Ser Glu Al - #a Ile Lys Gly Phe             #               510                                                           - Leu Val Lys Cys Cys Ala Tyr Asn Ser Leu Gl - #y Thr Ser Cys Glu             #               525                                                           - Thr Ile Leu Leu Asn Ser Pro Gly Pro Phe Pr - #o Phe Ile Gln Asp             #               540                                                           - Asn Ile Ser Phe Tyr Ala Thr Ile Gly Val Cy - #s Leu Leu Phe Ile             #               555                                                           - Val Val Leu Thr Leu Leu Ile Cys His Lys Ty - #r Lys Lys Gln Phe             #               570                                                           - Arg Tyr Glu Ser Gln Leu Gln Met Val Gln Va - #l Thr Gly Ser Ser             #               585                                                           - Asp Asn Glu Tyr Phe Tyr Val Asp Phe Arg Gl - #u Tyr Glu Tyr Asp             #               600                                                           - Leu Lys Trp Glu Phe Pro Arg Glu Asn Leu Gl - #u Phe Gly Lys Val             #               615                                                           - Leu Gly Ser Gly Ala Phe Gly Lys Val Met As - #n Ala Thr Ala Tyr             #               630                                                           - Gly Ile Ser Lys Thr Gly Val Ser Ile Gln Va - #l Ala Val Lys Met             #               645                                                           - Leu Lys Glu Lys Ala Asp Ser Ser Glu Arg Gl - #u Ala Leu Met Ser             #               660                                                           - Glu Leu Lys Met Met Thr Gln Leu Gly Ser Hi - #s Glu Asn Ile Val             #               675                                                           - Asn Leu Leu Gly Ala Cys Thr Leu Ser Gly Pr - #o Ile Tyr Leu Ile             #               690                                                           - Phe Glu Tyr Cys Cys Tyr Gly Asp Leu Leu As - #n Tyr Leu Arg Ser             #               705                                                           - Lys Arg Glu Lys Phe His Arg Thr Trp Thr Gl - #u Ile Phe Lys Glu             #               720                                                           - His Asn Phe Ser Phe Tyr Pro Thr Phe Gln Se - #r His Pro Asn Ser             #               735                                                           - Ser Met Pro Gly Ser Arg Glu Val Gln Ile Hi - #s Pro Asp Ser Asp             #               750                                                           - Gln Ile Ser Gly Leu His Gly Asn Ser Phe Hi - #s Ser Glu Asp Glu             #               765                                                           - Ile Glu Tyr Glu Asn Gln Lys Arg Leu Glu Gl - #u Glu Glu Asp Leu             #               780                                                           - Asn Val Leu Thr Phe Glu Asp Leu Leu Cys Ph - #e Ala Tyr Gln Val             #               795                                                           - Ala Lys Gly Met Glu Phe Leu Glu Phe Lys Se - #r Cys Val His Arg             #               810                                                           - Asp Leu Ala Ala Arg Asn Val Leu Val Thr Hi - #s Gly Lys Val Val             #               825                                                           - Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Il - #e Met Ser Asp Ser             #               840                                                           - Asn Tyr Val Val Arg Gly Asn Ala Arg Leu Pr - #o Val Lys Trp Met             #               855                                                           - Ala Pro Glu Ser Leu Phe Glu Gly Ile Tyr Th - #r Ile Lys Ser Asp             #               870                                                           - Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu Il - #e Phe Ser Leu Gly             #               885                                                           - Val Asn Pro Tyr Pro Gly Ile Pro Val Asp Al - #a Asn Phe Tyr Lys             #               900                                                           - Leu Ile Gln Asn Gly Phe Lys Met Asp Gln Pr - #o Phe Tyr Ala Thr             #               915                                                           - Glu Glu Ile Tyr Ile Ile Met Gln Ser Cys Tr - #p Ala Phe Asp Ser             #               930                                                           - Arg Lys Arg Pro Ser Phe Pro Asn Leu Thr Se - #r Phe Leu Gly Cys             #               945                                                           - Gln Leu Ala Asp Ala Glu Glu Ala Met Tyr Gl - #n Asn Val Asp Gly             #               960                                                           - Pro Val Ser Glu Cys Pro His Thr Tyr Gln As - #n Arg Arg Pro Phe             #               975                                                           - Ser Arg Glu Met Asp Leu Gly Leu Leu Ser Pr - #o Gln Ala Gln Val             #               990                                                           - Glu Asp Ser                                                                         993                                                                   __________________________________________________________________________

We claim:
 1. A method for enhancing proliferation or differentiation ofhematopoietic cells comprising contacting the hematopoietic cells withan effective amount of an agonist antibody which binds to theextracellular domain of flk2/flt3 and activates the tyrosine kinasedomain of flk2/flt3.
 2. The method of claim 1 wherein the agonistantibody is a monoclonal antibody.
 3. The method of claim 1 wherein theagonist antibody causes the differentiation or proliferation of myeloidand lymphoid blood cell lineages.
 4. The method of claim 1 furthercomprising contacting the hematopoietic cells with steel factor (SLF).5. A method for enhancing repopulation of mature blood cell lineages ina mammal comprising administering to the mammal a therapeuticallyeffective amount of an agonist antibody which binds to the extracellulardomain of flk2/flt3 and activates the tyrosine kinase domain offlk2/flt3.
 6. The method of claim 5 wherein the mammal has undergonechemotherapy, radiation therapy, or bone marrow transplantation therapy,or has suffered a decrease in blood cell lineages as a consequence ofhaving suffered hemorrhage or disease.
 7. The method of claim 3 whereinthe agonist antibody causes a synergistic increase in the proliferationor differentiation of primitive hematopoietic cells in combination withsteel factor (SLF).
 8. The method of claim 1 which enhances theproliferation or differentiation of primitive hematopoietic cells. 9.The method of claim 1 wherein the agonist antibody has the biologicalcharacteristics of the monoclonal antibody produced by the hybridomacell line deposited under American Type Culture Collection AccessionNumber ATCC HB 11,557.
 10. The method of claim 1 wherein the agonistantibody binds to the epitope to which the monoclonal antibody producedby the hybridoma cell line deposited under American Type CultureCollection Accession Number ATCC HB 11,557 binds.
 11. The method ofclaim 1 wherein the agonist antibody gives rise to an expansion in cellnumber of AA4⁺ Kit⁺ Flk-2⁺, AA4⁺ Sca⁺, AA4⁺ CD34⁺ Kit⁺, AA4⁺ CD34⁺Flk-2⁺, and LIN^(lo) CD34⁺ Flk-2⁺ cell populations in vitro.
 12. Themethod of claim 1 wherein the agonist antibody does not give rise to anexpansion in cell number of an AA4⁺ Sca⁻ cell population in vitro. 13.The method of claim 1 wherein the agonist antibody enhancesproliferation and differentiation of hematopoietic stem cells destinedto differentiate to more committed progenitor cells.
 14. The method ofclaim 1 wherein the agonist antibody comprises complementaritydetermining region (CDR) residues of a non-human antibody and frameworkregion residues of a human antibody.
 15. The method of claim 1 furthercomprising contacting the hematopoietic cells with a cytokine capable ofenhancing the proliferation or differentiation of hematopoietic cells.16. The method of claim 15 wherein the cytokine is selected from thegroup consisting of Epo, IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12, GM-CSF, G-CSF, M-CSF, SLF, LIF, TNF,lymphotoxin, flk2/flt3 ligand, kit-ligand, IGF-1 and γ-interferon. 17.The method of claim 1 wherein the hematopoietic cells are in a mammal.18. The method of claim 17 wherein the mammal is a human.
 19. The methodof claim 5 wherein the agonist antibody enhances proliferation anddifferentiation of myeloid and lymphoid blood cell lineages.
 20. Themethod of claim 19 wherein the agonist antibody causes a synergisticincrease in the proliferation or differentiation of primitivehematopoietic cells in combination with steel factor (SLF).